Lymphangiogenesis promoting factor-expressing fibroblasts and pharmaceutical compositions containing the same

Abstract

To provide lymphangiogenesis promoting factor-expressing fibroblasts and a pharmaceutical composition containing the same The following are the challenges. The present invention provides fibroblasts with high expression of ADM and / or HHEX as cells with lymphangiogenic potential. and the discovery of a method for treating fibrosis and lymphangiogenesis using a pharmaceutical composition containing the cells. Measures to improve hypotensive states, tissue fluid homeostasis, lipid and / or vitamin transport, and immune surveillance We have also discovered a means to regulate the immune system and treat organ failure. By culturing human fibroblasts using this medium, high expression of ADM and VCAM-1 was observed. We have discovered that improved adult-derived fibroblasts can be obtained, and provide fibroblasts with high ADM expression. We have discovered that these cells can be used to treat lung diseases.

Description

[Technical Field]

[0001] This invention relates to lymphangiogenesis-promoting fibroblasts, particularly those expressing specific genes and proteins (a drenomedullin (hereinafter also referred to as ADM) and / or Hematopoie tically-expressed homeobox protein (hereinafter referred to as HHE) Lympanic neoplasm-promoting fibroblasts expressing X, and a pharmaceutical compound containing the same. Regarding finished products. [Background technology]

[0002] Lymphatic vessels are a network of conduits that run parallel to the vascular system and are distributed throughout the body. Treatment and improvement of vascular disorders (Non-patent Literature 1), maintenance of homeostasis of tissue fluid in living organisms (Non-patent Literature 2), lipids Furthermore, it plays an important role in vitamin transport (Non-Patent Document 3) and the regulation of immune surveillance. (Non-patent document 4). In the heart, lymphatic vessels are responsible for maintaining normal cardiac output, and for the body fluids We are working to maintain a balance. In recent years, the effect of promoting lymphatic vessel formation (hereinafter referred to as lymphangiogenesis) has been observed in myocardial ischemia and myocardial infarction. It has been reported to be useful as a therapeutic target for subsequent recovery of cardiac output, and also for edema. It is also attracting attention as a therapeutic target for alleviating the condition (Non-Patent Document 1).

[0003] ADM is a cardioprotective peptide that plays an important role in the development of the cardiovascular and lymphatic systems. ADM has been reported to stabilize the barrier of lymphatic endothelial cells and promote lymphangiogenesis. This has been done (Non-Patent Document 5). In clinical practice, ADM is administered intravenously to patients with acute myocardial infarction. It has been reported that this can lead to significant improvements in the cardiovascular system (Non-Patent Literature 6). ).

[0004] HHEX is a direct transcriptional regulator of VEGFA, VEGFR1, and VEGFR2. In addition to being known to promote lymphangiogenesis via the VEGFC / FLT4 / PROX1 signaling pathway, It has been reported that this can be adjusted (Non-Patent Document 7).

[0005] Furthermore, interstitial pneumonia is caused by viruses such as the novel coronavirus infection (COVID-19). Risk factors include infection, aging, smoking, and diseases such as collagen diseases and sarcoidosis. As a result, the inflammatory response in the alveoli goes into a runaway state (cytokine storm), and fibrosis progresses within the alveoli. This refers to a condition in which the gas exchange function of the lungs is impaired (Non-Patent Literature 8). In particular, symptoms for which the causative factor cannot be identified are called idiopathic interstitial pneumonias (IIPs), and this disease This condition is designated as a "rare disease (specific disease)" in Japan. It affects 80-90% of IIPs patients. Idiopathic pulmonary fibrosis (IPF), which falls under this category, is particularly resistant to current treatments, and the condition after diagnosis is difficult to manage. The average survival time is 3-5 years, and the average survival time after acute exacerbation is less than 2 months, making it more probable than many other cancers. It is known as a disease with a poor prognosis. Against this backdrop, interstitial pneumonia and lung cancer, including IPF, are becoming more common. The development of treatments for vascular disease is progressing, and several drugs are being used clinically. For example, Pirfenidone slows the decline in forced ventilation (FVC) and 6-minute walk distance. It has been shown that it may improve mortality in certain patients. Nintedani b was also shown to slow the decline in FVC and reduce mortality. However, However, these treatment methods did not show improvement in respiratory symptoms, and the rate of acute exacerbations was not significantly improved. It has not improved. Therefore, it is clear that lung function will continue to deteriorate. Yes, they are. To overcome these problems, connective tissue growth factor, interleukin (IL)-4 and IL-13 antibodies, galectin-3 inhibitors, lysophosphatidic acid receptor antagonists, phos phoinositide 3 kinase pathway inhibitors, and dozens of novel therapeutic agents, including mesenchymal stem cells (MSCs), have been developed, and some of them are undergoing clinical trials. However, there is currently no effective treatment for the acute exacerbation of pulmonary fibrosis. In recent years, pulmonary fibrosis, like asthma and chronic obstructive pulmonary disease (COPD), has been reported to have activation of chronic and recurrent inflammatory responses as a pathophysiological risk factor. In a healthy lung, inflammatory cytokines control the inflammatory response of the lung against viral infection and injury, contributing to tissue repair and maintenance of homeostasis. However, in pulmonary fibrosis, inflammatory cytokines are overproduced, and the inflammatory response becomes a cytokine storm, inducing the formation of myofibroblast foci. As a result, excessive deposition of the extracellular matrix (ECM) occurs, leading to fibrosis

[0006] It has been shown (Non-Patent Document 9). It has been suggested that the induction of such fibrosis by excessive inflammatory responses involves structural and functional changes in lymphatic vessels (Non-Patent Document 10). In the lung, lymphatic vessels are composed of a single layer of lymphatic endothelial cells (LECs) with a discontinuous basement membrane and function as a transport route for antigens and antigen-presenting cells from peripheral tissues to lymph nodes and as a clearance of interstitial fluid balance. In fibrosis, lymphatic vessel damage occurs. For example, in a radiation-induced IPF animal model, a significant decrease in lymphatic vessel density has been reported to correlate with the induction of fibrosis (Non-Patent Document 11), and in a bleomycin-induced IPF animal model

[0007]

[0008]

[0009]

[0010] ]>

[0011]

[0012] In this case, it has been reported that platelet-derived growth factor receptor (PDGFR)-β-positive mural cells induce lymphatic vessel disorder in the lung by inhibiting the excretion of hyaluronic acid, which is an important molecule in lung injury and repair (Non-Patent Document 12). On the other hand, in human IPF, the lymphatic vessel density in lung tissue is increased, but the formed lymphatic vessels have structural and functional defects, and the localization of PDGFR-positive mural cells and the formation of intermittently disrupted lymphatic vessels are observed (Non-Patent Document 13). In addition, it has also been reported that overexpression of transforming growth factor (TGF)-β1 by fibrosis-inducing fibroblasts in wound healing inhibits the proliferation and function of lymphatic endothelial cells and selectively destroys lymphatic vessels (Non-Patent Document 14).

[0008] The present inventors have previously found cardiac-derived Vascular cell adhesion molecule-1 (VCAM-1, CD106)-positive fibroblasts that can efficiently induce lymphangiogenesis in the heart and greatly improve cardiac contractility in heart failure (Non-Patent Document 15), and have also found a method for producing VCAM-1-positive fibroblasts derived from the adult heart (Patent Document 1).

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

[0011] The present invention provides fibroblasts having lymphangiogenic ability and pharmaceutical compositions containing them. The challenge is to address the following issues. [Means for solving the problem]

[0012] The inventors of the present invention have conducted studies to solve the above problems and have brought TNF-α and IL-4 into contact. Fibroblasts significantly increase the gene expression levels of ADM and / or HHEX. The heading completes the invention.

[0013] In other words, the present invention includes the following first aspect (Invention A). (A1) A pharmaceutical composition comprising fibroblasts that highly express ADM and / or HHEX. (A2) The pharmaceutical composition according to (A1), wherein the fibroblasts are adult-derived fibroblasts. (A3) The gene expression levels of ADM and / or HHEX in the aforementioned cells are compared to those of control fibroblasts. The pharmaceutical composition described in (A1) or (A2) is 1.20 times or more higher in comparison to the above. . (A4) A pharmaceutical composition according to any of (A1) to (A3) for the treatment of fibrosis. (A5) To improve the condition in which lymphatic vessel formation is reduced, (A1) to (A4) Any of the pharmaceutical compositions described below. (A6) To improve impaired tissue fluid homeostasis, see (A1) to (A5) below. A pharmaceutical composition. (A7) A pharmaceutical composition according to any of (A1) to (A6) for reducing edema. (A8) To improve impaired lipid and / or vitamin transport capacity, (A1)~(A7) A pharmaceutical composition as described in any of the following. (A9) A pharmaceutical product described in any of (A1) to (A8) for activating the immune surveillance mechanism. composition. (A10) A pharmaceutical composition according to any one of (A1) to (A9), which is an injectable composition. (A11) The cells are constructed as a planar or three-dimensional cell tissue, (A A pharmaceutical composition as described in any of (1) to (A9). (A12) The fibroblasts are cardiac fibroblasts, of which (A1) to (A11) A pharmaceutical composition as described in any of the following.

[0014] Furthermore, the present invention includes the following second aspect (Invention B). (B1) A method for treating fibrosis in the subject, wherein ADM and / or HHEX are highly prevalent. A method comprising administering or transplanting a pharmaceutical composition containing present fibroblasts to a subject. (B2) A method to improve the condition in which lymphangiogenesis is reduced in the subject, A pharmaceutical composition containing fibroblasts with high expression of DM and / or HHEX is administered to or transplanted into the subject. A method that includes doing something. (B3) A method for improving impaired tissue fluid homeostasis in the subject, comprising ADM and / or This includes administering or transplanting a pharmaceutical composition containing fibroblasts with high HHEX expression to the subject. Hmm, a method. (B4) A method for reducing edema in the subject, wherein ADM and / or HHEX high expression A method comprising administering or transplanting a pharmaceutical composition containing fibroblasts to a subject. (B5) A method for improving impaired lipid and / or vitamin transport capacity in the subject. , administering a pharmaceutical composition containing fibroblasts that highly express ADM and / or HHEX to the subject or A method that includes transplantation. (B6) A method for activating the immune surveillance mechanism in a subject, wherein ADM and / or HH A method comprising administering or transplanting a pharmaceutical composition containing fibroblasts with high EX expression to the subject. Law. (B7) A method for treating organ failure in the subject, wherein ADM and / or HHEX levels are elevated. A method comprising administering or transplanting a pharmaceutical composition containing expressed fibroblasts to a subject.

[0015] Furthermore, the present invention includes the following third aspect (Invention C). (C1) A pharmaceutical composition containing fibroblasts with high ADM and / or HHEX expression, for use in organs with dysfunction Administer to the tissue and / or surrounding area, or to fibrotic tissue and / or surrounding area. For use as an injectable agent. (C2) A pharmaceutical composition containing fibroblasts with high ADM and / or HHEX expression, for organ failure To be transplanted into the tissue and / or surrounding area, or into fibrous tissue and / or surrounding area. Use as transplant material.

[0016] The present invention includes the following fourth aspect (Invention D). (D1) A method for producing fibroblasts that highly express ADM, Contacting fibroblasts with TNF-α and IL-4, and The fibroblasts that have been in contact with the cells are cultured under conditions that can lead to high expression of ADM. A method for producing fibroblasts that highly express ADM, including (D2) The method according to (D1), wherein the fibroblasts are highly expressive of VEGF-C. (D3) The method according to (D1) or (D2), wherein the fibroblasts are CD106 positive. . (D4) Select or enrich fibroblasts that highly express ADM using an anti-CD106 antibody. The method described in (D3), including the method described in (D3). (D5) The fibroblasts are lung-derived fibroblasts, or any of (D1) to (D4) Method of description. ADM high obtained by the manufacturing method described in any of (D6), (D1), to (D5) Fibroblasts expressing this trait. A population of fibroblasts, including the cells described in (D7) and (D6).

[0017] The present invention includes the following fifth aspect (Invention E). (E1) Fibroblasts with high ADM expression. (E2) Fibroblasts described in (E1) that have high VEGF-C expression. (E3) Fibroblasts described in (E1) or (E2) that are positive for CD106. (E4) The gene expression levels of VEGF-C and / or ADM in the cells are compared to control fibroblasts. Compared to cells, any of (E1) to (E3) is 1.20 times or higher. The cells described. (E5) The fibroblasts are lung-derived fibroblasts, or any of (E1) to (E4) The cells described. A population of fibroblasts containing any of the cells described in (E6), (E1), or (E5). (E7) A cell population including lung-derived fibroblasts that are positive for CD106, The proportion of lung-derived fibroblasts that are CD106-positive (based on cell count) in the aforementioned cell population A cell population that accounts for more than 18.01% of all fibroblasts contained within.

[0018] The present invention includes the following sixth aspect (Invention F). Cells described in any of (F1), (D6), and (E1) to (E5), or (D7) and (E A pharmaceutical composition comprising the cell population described in any of 6) to (E7). (F2) The pharmaceutical composition described in (F1) for treating lung disease. (F3) The pharmaceutical composition according to (F2), wherein the lung disease is pulmonary fibrosis or interstitial pneumonia. (F4) The medicinal combination described in (F1) for improving the condition in which lymphangiogenesis is impaired. Finished product. (F5) A pharmaceutical composition according to any one of (F1) to (F4), which is an injectable composition. (F6) The cells or the cell population are constructed as a cell tissue in a planar or three-dimensional shape. A pharmaceutical composition as described in any of (F1) to (F4).

[0019] The present invention includes the following seventh aspect (Invention G). (G1) A method for treating lung disease in the subject, (D6) and (E1)~(E5 Cells described in any of the above, or a collection of cells described in any of (D7) and (E6) to (E7). A method comprising administering or implanting the pharmaceutical composition described in (F1) to a subject. (G2) The method according to (G1), wherein the lung disease is pulmonary fibrosis or interstitial pneumonia. (G3) A method to improve the condition in which lymphangiogenesis is reduced in the subject, Cells described in D6) and any of (E1) to (E5), (D7) and (E6) to (E7 The cell population described in any of the above or the pharmaceutical composition described in (F1) is administered to the subject or transferred A method that includes planting.

[0020] The present invention includes the following eighth aspect (Invention H). Cells described in any of (H1)(D6) and (E1)~(E5), (D7) and (E6 Lung tissue and the cell population described in any of (E7) or the pharmaceutical composition described in (F1) Use as an injectable preparation for administration to the and / or surrounding area. Cells described in (H2)(D6) and any of (E1)~(E5), (D7) and (E6 Lung tissue and the cell population described in any of (E7) or the pharmaceutical composition described in (F1) Use as a transplant material for transplantation to the and / or surrounding areas. [Effects of the Invention]

[0021] The present invention provides a pharmaceutical composition containing fibroblasts with lymphangiogenic ability, namely ADM. and / or a pharmaceutical composition containing HHEX-highly expressing cells can be provided, and the pharmaceutical composition Methods for treating fibrosis using materials, methods for improving impaired lymphangiogenesis, and constant tissue fluid It provides means to regulate stasis, lipid and / or vitamin transport, immune surveillance, and treat organ failure. We were able to do this. In addition, we provided fibroblasts that highly express ADM, and using these cells We were able to provide a means to treat lung diseases. [Brief explanation of the drawing]

[0022] [Figure 1] This figure shows the characteristics of adult-derived cardiac fibroblasts under various culture conditions. A is a micrograph of cultured cardiac fibroblasts (scale bar: 500 μm). Untreated (NT) represents untreated cardiac fibroblasts without cytokine supplementation, and +CKs represents cardiac fibroblasts supplemented with TNF-α and IL-4. B is a flow cytometry analysis. The scatter plot shows CD90 expression on the horizontal axis and CD106 expression on the vertical axis. The histograms show CD90 expression and CD106 expression, respectively. [Figure 2]This figure shows the expression levels of lymphangiogenesis genes in adult cardiac fibroblasts under various culture conditions. A shows the results of real-time quantitative RT-PCR (RT-qPCR) analysis indicating ADM mRNA expression. In all groups, ADM gene expression levels in fibroblasts were standardized by β-Actin gene expression levels, and the difference in gene expression levels for each condition is expressed as a Fold increase. NT represents untreated cardiac fibroblasts, and +CKs represents cardiac fibroblasts treated with TNF-α and IL-4. B shows the results of RT-qPCR analysis indicating HHEX mRNA expression levels. Similar to ADM, HHEX gene expression levels in all groups were standardized by β-Actin and expressed as a Fold increase (**P<0.01 vs. NT). [Figure 3] This figure illustrates the lymphangiogenic effect of human adult-derived cardiac fibroblasts. Figure A shows a photograph of the vascularization effect of various human cardiac fibroblasts on cardiac lymphatic vessels, as observed with a fluorescence microscope. The capillary-like structures represent vascular endothelial (VE)-cadherin-positive cells and refer to vessels formed by lymphatic endothelial cells recovered from cardiac-derived cells. Other cellular regions are indicated with Vimentin to represent various cardiac fibroblasts. The nucleus is indicated with Hoechst33258 (10x magnification). NT represents untreated cardiac fibroblasts, and +CKs represents cardiac fibroblasts treated with TNF-α and IL-4. Figure B shows the quantitative evaluation of the fluorescence microscope images. NT is set to 1, and the result is expressed as Fold increase (**P<0.01 vs. NT, *P<0.05 vs. NT, N=12). [Figure 4]This figure shows flow cytometry analysis of various fibroblast cells. The upper panel shows the CD90 and CD106 expression levels in human adult-derived cardiac fibroblasts (A-HCF), adult-derived cardiac fibroblasts cultured for 72 hours with the addition of TNF-α and IL-4 (uA-HCF), and CD90-positive adult-derived cardiac fibroblasts (uA90-HCF) cultured for 72 hours with the addition of TNF-α and IL-4, followed by cell sorting based on CD90. The lower panel shows the CD90 and CD106 expression levels in human fetal-derived cardiac fibroblasts (F-HCF), CD106-negative fetal-derived cardiac fibroblasts (F-VNCF), and CD106-positive fetal-derived cardiac fibroblasts (F-VCF). [Figure 5] This figure illustrates the therapeutic effect of human adult-derived cardiac fibroblasts on fibrosis. A is a micrograph showing Sirius Red (SR) stained cardiac tissue from chronic heart failure rats administered with various adult-derived cardiac fibroblasts (+A-HCFs, +uA-HCFs, +uA90-HCFs). B is a figure showing the quantitative evaluation of fibrotic areas in chronic heart failure rat cardiac tissue administered with various adult-derived cardiac fibroblasts (N=4). C is a micrograph showing Sirius Red stained cardiac tissue from chronic heart failure rats administered with fetal-derived cardiac fibroblasts (+F-VCF). Figure D shows the quantitative evaluation of fibrotic areas in the cardiac tissue of chronic heart failure rats administered with fetal-derived cardiac fibroblasts (+F-VCF) (NS=not significant, N=4, **P<0.01 vs. Control, *P<0.05 vs. Control, ††P<0.01 vs. Sham, †P<0.05 vs. Sham, ‡P<0.05 vs. +A-HCFs). [Figure 6]This figure illustrates the therapeutic effect of human adult-derived cardiac fibroblasts on fibrosis. A is a micrograph showing hematoxylin-eosin (HE) stained cardiac tissue from chronic heart failure rats administered various adult-derived cardiac fibroblasts (+A-HCFs, +uA-HCFs, +uA90-HCFs). B is a magnified micrograph of the parts a-l described in A. C is a table showing the pathological qualitative scoring. The degree of pathological change was qualitatively evaluated as no change (score 0), mild (score 1), moderate (score 2), and severe (score 3). The scoring items were inflammation cell aggregation, proliferation of fibroblasts, fibrosis, lime deposition, and thinning of the left ventricular wall. [Figure 7] This image demonstrates the lymphangiogenic effect of human adult-derived cardiac fibroblasts on myocardium. It is a micrograph (in situ hybridization) showing Lyve-1 stained images of chronic heart failure rat cardiac tissue administered with adult-derived cardiac fibroblasts (+A-HCFs, +uA-HCFs, +uA90-HCFs). Each panel in the right column is a magnified view of the area within the square frame in the respective panel in the left column. [Figure 8] This figure shows the localization of lymphatic endothelial cells in rat hearts. The images are micrographs representing immunohistochemical staining of cardiac tissue from chronic heart failure model rats administered adult-derived cardiac fibroblasts (+A-HCFs, +uA-HCFs, +uA90-HCFs). Low and high magnification images are shown for +uA90-HCFs. Cardiac structures are shown with cTnT, central tubular structures with Prox-1 or VEGFR3, and nuclei with DAPI. [Figure 9]This figure shows the morphology and lymphangiogenic capacity of human adult-derived lung fibroblasts. A is a micrograph (5× magnification) showing a bright-field image of human lung fibroblasts. B is a micrograph (5× magnification) showing a bright-field image of human lung fibroblasts (Control) and human lung fibroblasts (+Factor-X) cultured for 24 hours with the addition of TNF-α (50 ng / mL) and IL-4 (2 ng / mL). C and D show the percentage of CD90 (fibroblast marker) and CD106 (VCAM-1) positive cells of various human lung fibroblasts by flow cytometry (FCM) analysis (N=3, **P<0.01, Student's t-test, NS indicates no significant difference). E shows a relative comparison of vascular endothelial growth factor (VEGF)-C and Adrenomedullin (ADM) gene expression levels of various human lung fibroblasts by RT-qPCR analysis. The gene expression levels of the control group were set to 1 (N=3, **P<0.01, *P<0.05, Student's t-test). [Figure 10] This figure shows the morphology of lymphatic endothelial cells recovered from human lung-derived cells and the lymphangiogenic effect of human adult-derived lung fibroblasts. A is a micrograph (5× magnification) showing a bright-field image of human microvascular endothelial cells. B shows the percentage of vascular endothelial (VE)-Cadherin and podoplanin (PDPN) positive cells of human microvascular endothelial cells by FCM analysis (in each graph, the left peak shows isotype control stained samples, and the right peak shows various antibody stained samples). C is a photograph showing the vascularization effect of various human lung fibroblasts on pulmonary lymphatic vessels by fluorescence microscopy. The capillary-like structures indicate VE-Cadherin positive cells and refer to vessels formed by lymphatic endothelial cells recovered from lung-derived cells. Other cell regions are indicated by Vimentin to show various lung fibroblasts. The nuclei are indicated by Hoechst33258 (10× magnification). [Figure 11]This figure shows the morphology and lymphangiogenic capacity of adult mouse lung fibroblasts. A is a micrograph (5× magnification) showing a bright-field image of mouse lung fibroblasts. B shows the percentage of VCAM-1 positive cells in mouse lung fibroblasts by FCM analysis (in each graph, the left peak represents the isotype control stained sample, and the right peak represents the various antibody stained samples). C shows a relative comparison of VEGF-C and ADM gene expression levels in various mouse lung fibroblasts by RT-qPCR analysis. The gene expression level of the control is set to 1 (N=3, **P<0.01, *P<0.05, Student's t-test). [Figure 12] This figure shows the therapeutic effect of adult-derived pulmonary fibroblasts (VPF) with high ADM expression and enhanced VCAM-1 expression on interstitial pneumonia and pulmonary fibrosis. A shows the animal experiment design. Cells were administered to disease model mice 7 days after administration of bleomycin solution, and the mice were sacrificially killed 4 weeks later to collect lung tissue. B shows the survival analysis of each group by Kaplan-Meier method. The dots indicate the censoring point. C is a micrograph representing the Sirius Red stained image of each group. [Figure 13] This figure shows the therapeutic effects of various fibroblasts two weeks after administration. A shows the survival analysis. The PF administration group (+mPF) consisted of n=7, the mVPF administration group (+mVPF) consisted of n=5, and the physiological saline administration group (Ctrl) consisted of n=11. The p-value was calculated using the log-rank (Mantel-Cox) test. B shows micrographs of the fibrotic areas and alveolar structures of the lungs in each group. Lung tissue collected 14 days after cell administration was stained with Masson's Trichrome. BLM shows lung tissue on day 0 after cell administration, and Ctrl shows lung tissue from the physiological saline administration group (scale bar: 500 μm). C represents the quantitative assessment of pulmonary fibrosis in each group (BLM: n=5, Ctrl: n=6, +mPF: n=4, +mVPF: n=5, *P<0.05, ***P=0.0006, ns indicates no statistically significant difference, ordinary one-way ANOVA with Tukey's multiple comparisons test). [Figure 14]This figure shows the cellular characteristics of rPF and rVPF recovered by primary. A is a micrograph showing the appearance of rPF at each passage. PN refers to the Passage number. B is a photograph showing bright-field imaging of rPF and rVPF. C shows the flow cytometry analysis. [Figure 15] This figure shows the therapeutic effects of various fibroblasts two weeks after administration. A is a micrograph showing the fibrotic area and alveolar structure of the lungs in each group. Lung tissue collected on day 0 or 14 after cell administration was stained with Masson's Trichrome. BLM shows the lung tissue on day 0 after cell administration, and Ctrl shows the lung tissue of the group administered physiological saline. B is a graph showing the quantitative evaluation of the fibrotic area of ​​the lungs in each group (BLM: n=5, Ctrl: n=6, +rPF: n=7, +rVPF: n=8, **P<0.01, ***P=0.0001, ****P<0.0001, ns indicates no significant difference, calculated by ordinary one-way ANOVA with Tukey's multiple comparisons test). [Figure 16] This shows the percentage of CD90 (fibroblast marker) and CD106 (VCAM-1) positive cells in various human lung fibroblasts, as determined by flow cytometry (FCM) analysis (N=5, ****P<0.0001, ns indicates no significant difference, calculated by 2-way ANOVA with Tukey's multiple comparisons test). hPF refers to human lung fibroblasts, and hPF+CKs refers to human lung fibroblasts cultured for 72 hours with the addition of TNF-α (50 ng / mL) and IL-4 (2 ng / mL). hVPF refers to hPF+CKs sorted using CD106 as an indicator. [Modes for carrying out the invention]

[0023] The present invention will be described in detail below using specific embodiments, but the present invention is a tool It is not limited to physical embodiments.

[0024] <Pharmaceutical composition containing fibroblasts with high expression of ADM and / or HHEX> One embodiment of the first invention is a pharmaceutical composition containing fibroblasts with high expression of ADM and / or HHEX.

[0025] There is no limitation on the origin of the fibroblasts, and pluripotent stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and Muse cells, as well as adult stem cells such as mesenchymal stem cells, may be differentiated and used. Also, primary cells collected from animals (including humans) may be used as well as immortalized cells. Examples of the organs from which the fibroblasts are derived include heart-derived, kidney-derived, skin-derived, lung-derived, etc. and the like. The fibroblasts are not particularly limited, but are preferably derived from adults. The fibroblasts from which the fibroblasts with high expression of ADM and / or HHEX are derived may originally express a certain amount of ADM and / or HHEX, or may not express it at all. In this embodiment, the fibroblasts may be cells expressing cell surface markers expressed by normal fibroblasts, and further, according to the method for producing the cells of the present invention described later, cells in which additional other markers are expressed or some markers are not expressed may also be used.

[0026] When ADM is highly expressed, it is not particularly limited. For example, when the mRNA expression level is measured by RT-q PCR, when the mRNA expression level of the control fibroblasts is set to 1, the mRNA expression level of ADM in the fibroblasts in the pharmaceutical composition of this embodiment is 1.20 times or more, 1.40 times or more, 1.50 times or more, 1.60 times or more, 1.70 times or more, 1.80 times or more, 1.90 times or more, 2.00 times or more, 2.30 times or more, 3.00 times or more, 4.00 times or more, 5.00 times or more, 6.00 times or more, 7.00 times or more, 8.00 times or more, 9.00 times or more, It is preferable that the increase is 10.0 times or more, or a multiple of 10.0 times or more. Note that the mRNA expression level of control fibroblasts refers to the mRNA expression level of untreated fibroblasts. This could be the NA expression level, or the average mRNA expression level of the entire cell body after treatment. . Furthermore, when protein expression levels were measured using flow cytometry, ADM positivity The proportion of fibroblasts (hereinafter also referred to as ADM+) is 0.1% or more based on the cell number. It may be present, it may be 1% or more, it may be 2% or more, it may be 4% or more, 5 It may be % or more, 10% or more, 20% or more, 30% or more It's fine if it's 40% or more, fine if it's 50% or more, fine if it's 60% or more. It may be 70% or more, 80% or more, 90% or more, 95 It may be % or more, 98% or more, or 99% or more.

[0027] High HHEX expression is not particularly limited, but for example, it can mean that mRNA expression levels are RT- When measured by qPCR, if the mRNA expression level of control fibroblasts is set to 1, then the actual The mRNA expression level of HHEX in fibroblasts in the pharmaceutical composition in application form is 1.20 times or more. Above, 1.40x or more, 1.50x or more, 1.60x or more, 1.70x or more, 1.80x or more Above, 1.90x or more, 2.00x or more, 2.58x or more, 3.00x or more, 4.00x or more Above, 5.00x or more, 6.00x or more, 7.00x or more, 8.00x or more, 9.00x or more Preferably, the increase is 10.0 times or more, or a multiple of 10.0 times or more. Note that the mRNA expression level of control fibroblasts refers to the mRNA expression level of untreated fibroblasts. This could be the NA expression level, or the average mRNA expression level of the entire cell body after treatment. . Furthermore, when protein expression levels were measured using flow cytometry, HHEX positive The proportion of fibroblasts (hereinafter also referred to as HHEX+) was 0.03% each, based on cell count. It may be more than or equal to 0.1% or more, it may be more than or equal to 1% or more, and it may be more than or equal to 2% or more. It is fine if it is 4% or more, fine if it is 5% or more, fine if it is 10% or more, and 20 It may be % or more, 30% or more, 40% or more, 50% or more It's fine if it's 60% or more, fine if it's 70% or more, fine if it's 80% or more. It may be 90% or more, 95% or more, 98% or more, 99 It can be % or more.

[0028] In this embodiment, the lymphangiogenesis-promoting factor is preferably ADM or HHEX It refers to genes or proteins. The nucleotide and amino acid sequences of lymphangiogenesis-promoting factors are not particularly limited, but for example, ADM is a gene having the base sequence represented by Sequence ID No. 1, or 90% of that base sequence. The above refers to genes having 95% or more or 98% or more identical base sequences, and the sequence number A complementary sequence to all or part of the base sequence represented by 1 under stringent conditions A gene that encodes a protein that is hybridized and has lymphangiogenic ability. Also, HHEX is a gene having the base sequence represented by Sequence ID No. 2, or that base sequence A gene having the same base sequence as 90% or more, 95% or more, or 98% or more, The complementary sequence and stringent sequence for all or part of the base sequence represented by Sequence ID No. 2 A gene that hybridizes under the same conditions and encodes a protein with lymphangiogenic ability. That's fine.

[0029] The fibroblasts in the pharmaceutical composition of this embodiment express CD106 as a cell surface marker. It's okay to do so.

[0030] The fibroblasts in the pharmaceutical composition of this embodiment exhibit high expression of ADM and / or HHEX as described above. A ramen dish containing a population of fibroblasts, including fibroblasts. In this embodiment, a method for producing fibroblasts with high ADM and / or HHEX expression, as described later, is used. The percentage of CD106-positive cells in a fibroblast population, including fibroblasts produced by [the company / organization], is It's fine if it's high, but the reverse isn't necessarily true.

[0031] (Preparation of fibroblasts) In the preparation of fibroblasts, the origin of the fibroblasts is not particularly limited, as already mentioned. On the other hand, the cells may be of the patient's own origin, for example, the heart of a patient suffering from heart disease. Cardiac fibroblasts isolated from tissue, and kidney cells isolated by differentiating adult (somatic) stem cells from patients. Prepare visceral fibroblasts. Also, differentiate pluripotent stem cells such as iPS cells derived from the patient's own cells. Furthermore, harvested fibroblasts may also be used. In addition, cells other than the self may be used, for example If heart cells are provided, they can be derived from donor heart tissue or from heart tissue created using animals, etc. Isolated cardiac fibroblasts and cardiac fibers isolated by differentiating adult (somatic) stem cells from donor Prepare blast cells. Also, differentiate pluripotent stem cells such as donor-derived iPS cells and ES cells. Then, the collected fibroblast cells were removed.

[0032] (Isolation of fibroblasts) Prepared fibroblasts are typically supplied by enzymes such as dispase and collagenase. It can be separated by processing. Separation is done by enzymes such as dispase and collagenase. In addition to being carried out, if necessary separation is possible, other processes, such as mechanical processes, may also be performed. That's fine.

[0033] (Method for producing fibroblasts that highly express ADM and / or HHEX) Contacting fibroblasts with TNF-α and IL-4, and the contacted fibroblasts This includes culturing cells under conditions that may result in high expression of ADM and / or HHEX. be.

[0034] The method of contact between fibroblasts and TNF-α and IL-4 involves high levels of ADM and / or HHEX It is not particularly limited unless the induction of existing fibroblasts is significantly impaired, and typically fibroblasts This involves adding TNF-α and IL-4 to the culture medium, but is not limited to this. -α and IL-4 may be added individually or in multiple types. In this case, each may be brought into contact with the fibroblasts separately, or they may be brought into contact simultaneously. For example, TNF-α and IL-4 are mixed with water, culture medium, serum, or dimethyl sulfoxide (DMS). It may be added to the culture medium after being dissolved in (O).

[0035] Since fibroblasts can be cultured in a medium supplemented with TNF-α and IL-4, Fibroblasts can be cultured while maintaining the expression levels of ADM and / or HHEX in the cells. Therefore, to produce fibroblasts with high ADM and / or HHEX expression levels... It is possible.

[0036] When adding TNF-α and IL-4 to the culture medium (mL), the amount of TNF-α added is not particularly limited. Although not fixed, it is usually 0.1 ng / mL or higher, and may be 0.5 ng / mL or higher. It may be 1 ng / mL or more, 10 ng / mL or more, and 50 ng / mL or more. It may be. There is no particular upper limit, and it is usually 500 ng / mL or less, and 100 ng / It may be less than or equal to mL. Furthermore, while there are no particular restrictions on the amount of IL-4 added, it is usually 0.1 ng / mL or more. It may be 0.5 ng / mL or higher, it may be 1 ng / mL or higher, and it may be 2 ng / mL or higher. It is acceptable. There is no particular upper limit, but it is usually 10 ng / mL or less, and 5 ng / mL or less. It may be 1 ng / mL or less. The ratio (by weight) of TNF-α to IL-4 to be added is typically 100:1. It can be 00:1~1:1, or 50000:1~10:1. Also, 1:1~1 It is 10000, and can be anywhere from 1:10 to 1:50000.

[0037] Further culturing of fibroblasts contacted with TNF-α and IL-4 allows for the development of ADM and / Alternatively, HHEX+ fibroblasts can be produced. Fibroblast culture is performed under conditions that the fibroblasts may become ADM and / or HHEX positive. The method is not particularly limited and may be carried out using known cell culture methods.

[0038] The culture medium used for culturing can be appropriately set depending on the type of cells being cultured, for example, DMEM, α-MEM, RPMI-1640, HFDM-1(+), etc. are available. Nutrients such as FCS and FBS, growth factors, cytokines, antibiotics, etc. are added to the culture medium. Furthermore, the cells may be cultured in a medium containing TNF-α and IL-4. The culture period is until the cultured cells reach the desired number of cells and / or until the cultured cells reach the desired number of cells. The culture time and number of culture days can be set appropriately depending on the purpose, such as until the function is acquired. For example, 1 Hours: 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 21 hours, 1 day, 2 days, 3 days, 4 days, 5 days, Periods such as 6 days, 7 days, 8 days, 9 days, 10 days, 2 weeks, 1 month, 2 months, 3 months, 6 months, etc. I can give it to you. The culture temperature can be set appropriately according to the type of cells being cultured, but for example, above 10°C. , may be 15℃ or higher, 20℃ or higher, 25℃ or higher, 30℃ or higher, and also 60℃ or lower, 5 The temperature may be 5°C or lower, 50°C or lower, 45°C or lower, or 40°C or lower. The culture process may be repeated multiple times. For example, the purity of the desired fibroblasts can be improved by selection or concentration. This allows for cultivation to be performed each time.

[0039] In the production of fibroblasts, an anti-CD106 antibody is further used to produce ADM and / or HHE. The method may include selecting or enriching fibroblasts that highly express X. This is not particularly limited and can be done by methods known to those skilled in the art, for example, human CD106(V Primary immunohistochemical staining was performed using CAM-1)-biotin (Miltenyi Biotec). Secondary immunostaining was performed using anti-biotin microbeads (Miltenyi Biotec). The cells were then collected using autoMACS (Miltenyi Biotec). This can be done. Furthermore, selection or concentration can be performed at any time. Anti-CD106 antibodies can be any known type; commercially available products can be used. can. By selecting or enriching with an anti-CD106 antibody, CD10 in a fibroblast population It can increase the percentage of 6-positive fibroblasts, and furthermore, ADM and / or HHEX+ The proportion of fibroblasts can be increased.

[0040] Cultured fibroblasts can be harvested by detaching them using proteases such as trypsin. You may collect them, but use a temperature-responsive culture dish to detach the cells due to temperature changes, and then... The cells may be collected, or they may be detached and collected using an instrument such as a cell scraper. .

[0041] By administering or implanting the pharmaceutical composition of this embodiment into the body, lymphatic vessel regeneration occurs in the body. It can promote life. Therefore, although not particularly limited, for example, damage to organ tissue, To improve conditions in which lymphangiogenesis is reduced due to defects or dysfunction, and tissue To improve impaired fluid homeostasis and impaired lipid and / or vitamin transport capacity, and immune surveillance. It can activate the mechanism. It can also be administered to patients with organ failure. It may also be possible to treat organ failure by doing so. Organ failure refers to a condition in which an organ does not perform its expected function and / or is clinically diagnosed. This refers to organ dysfunction that is so severe that normal homeostasis cannot be maintained without intervention. These include, but are not limited to, the heart, kidneys, adrenal glands, thyroid gland, parathyroid gland, vocal cords, and chest. Glands, cerebrum, cerebellum, brainstem, spinal cord, peripheral nerves, skin, muscles, eyeballs, tongue, esophagus, stomach, gallbladder, bile ducts , pancreas, pancreatic duct, liver, spleen, small intestine, large intestine, rectum, anus, trachea, bronchi, lungs, bladder, ureter, Prostate, artery, vein, lymphatic vessel, lymph node, bone, bone marrow, cartilage, tendon, tooth, testicle, vas deferens, penis These include the uterus, ovaries, fallopian tubes, and vagina. For example, organ failure in the heart or kidneys is... This can also refer to a state in which the function of an organ is reduced compared to its normal state, and in particular, when the function of an organ is normal It may also refer to a state where the level has decreased to 30% or less compared to normal. By administering or implanting the pharmaceutical composition of this embodiment into a living body, fibrosis can be treated or improved. It can be treated. The site of onset of fibrosis is not particularly limited; for example, it can occur in the heart, lungs, kidneys, etc. These are some examples.

[0042] The method of administering or implanting the pharmaceutical composition of this embodiment into a living organism is not particularly limited, but for example... It can be administered by injection. The pharmaceutical composition is administered to the insufficient organ or the area surrounding its infarction (for example, (Submembrane, etc.) or the damaged area, fibrotic area, or surrounding area in organs where fibrosis has developed. Direct administration or transplantation to the affected area can improve the above-mentioned disease conditions and treat organ failure. Furthermore, the pharmaceutical composition is used in veins, arteries, lymph nodes, lymphatic vessels, bone marrow, subcutaneous tissue, corpus cavernosum, and abdominal cavity. It may be administered internally, intramuscularly, intrathecally, or into the joint cavity. For administration into veins, arteries, or lymphatic vessels, use intravascular administration. This may be done by injection into the cerebral cortex, or by injection via catheter, or by other known You may use the following method. The method of injection is not particularly limited; known injection techniques such as needle injection and needle-free injection may be applied. It is possible to use known catheter methods for injection, and there are no particular limitations. do not have.

[0043] Furthermore, the pharmaceutical composition of this embodiment is not particularly limited, but is suitable for use in living organisms as an artificial organ material. It may also be used as an artificial organ material, for example, the cells may be arranged in a planar or three-dimensional cell group. It may also be constructed as a fabric. This planar or three-dimensional cell tissue consists of fibroblasts. It may be a culture of fibroblasts alone, or a co-culture of fibroblasts with other cells, such as kidney cells. The cell tissue may be in a planar or three-dimensional form. , but not limited to, cell sheets, cell fibers, and those formed by 3D printing. Examples include organizations that have been established. In this embodiment, the size and shape of the planar or three-dimensional cell tissue are determined by necrosis, injury, or Applicable to fibrotic organ tissue and / or its surrounding areas and / or lesion areas, in particular limited to the extent applicable. It is not specified. Furthermore, "planar" can refer to a single layer or multiple layers. Furthermore, conditions such as the culture medium composition, temperature, and humidity in cell culture are determined by ADM and / or HH EX+ fibroblasts can be cultured, and planar or three-dimensional cell tissues can be constructed. It is not particularly limited to the term. Such planar or three-dimensional cell tissues are necrotic, damaged, or fibrotic organ tissues and / or By applying it to the surrounding area, or as an artificial organ, it can be used to treat necrotic, damaged, or fibrotic organ groups. By replacing tissue and / or surrounding areas, it improves conditions in which lymphatic vessel formation is impaired. This involves improving the homeostasis of tissue fluid and impaired lipid and / or vitamin transport. This includes activating immune surveillance mechanisms, treating organ failure, and treating fibrosis. It can be improved.

[0044] The proportion of ADM and / or HHEX+ fibroblasts contained in the pharmaceutical composition is the amount administered to the patient. Alternatively, it may be appropriately determined based on the type of transplantation, etc., and may contain a therapeutically effective amount of ADM and / Alternatively, it is sufficient if it contains HHEX+ fibroblasts. If the pharmaceutical composition contains other fibroblasts, for example, fibroblasts contained in the pharmaceutical composition The proportion of ADM and / or HHEX+ fibroblasts relative to the total amount should be 0.03% or less by cell number. It may be above, it may be 0.1% or more, it may be 1% or more, and it may be 2% or more Often, 4% or more is fine, 5% or more is fine, 10% or more is fine, 20% It is acceptable if it is above 30%, acceptable if it is above 40%, acceptable if it is above 50% It's often the case that it's good if it's 60% or more, 70% or more, 80% or more. , may be 90% or more, may be 95% or more, may be 98% or more, and 99% That's all. Furthermore, when human-derived fibroblasts are used, ADM and / or H contained in the pharmaceutical composition may be present. In terms of HEX+ fibroblast count, control fibroblasts showed ADM and / or HHEX expression. ADM and / or HHEX expression levels in ADM and / or HHEX+ fibroblasts relative to the amount is 1.20 times or more, 1.40 times or more, 1.50 times or more, 1.60 times or more, 1.70 times or more Above, 1.80x or more, 1.90x or more, 2.00x or more, 3.00x or more, 4.00x or more Above, 5.00x or more, 6.00x or more, 7.00x or more, 8.00x or more, 9.00x or more The above can be 10.0 times or more. Furthermore, the number of ADM and / or HHEX+ fibroblasts contained in the pharmaceutical composition is, for example, 1. 0 x 10 2 cells / mL or more, 1.0×10 3 cells / mL or more, 1.0×10 4 cells / mL or more, 1.0×10 5cells / mL or more, 1.0×10 6 cel ls / mL or more, 5.0×10 6 cells / mL or more, 1.0×10 7 cells / m It can be L or more. The number of CD106-positive fibroblasts contained in the injectable composition is the disease The dosage may be further increased or decreased depending on the severity of the illness.

[0045] The method for confirming the therapeutic and / or improvement effects of the pharmaceutical composition is not particularly limited. For example, lymphangiogenesis increases after administration of the composition or transplantation compared to before administration of the composition or transplantation. Furthermore, the function of the organs is restored after administration of the composition or transplantation compared to before administration of the composition or transplantation. This involves comparing the degree of organ damage and line formation after administration or transplantation of the composition with the degree of organ damage after administration or transplantation of the composition. Examples include a reduction in the degree of phytochemistry.

[0046] The pharmaceutical composition may contain other components that are physiologically acceptable as a pharmaceutical composition. Other components include physiological saline, cell preservation solution, cell culture medium, hydrogel, and cells. Examples include outer matrix and cryopreservation solution.

[0047] Another embodiment of the first invention is the above-mentioned ADM and / or HHEX high-expression cells. By administering or implanting the pharmaceutical composition containing the above into fibrotic tissue and / or surrounding areas, This may be a method to treat or improve fibrosis. Furthermore, another embodiment of the first invention is the high expression of ADM and / or HHEX. The administration or transplantation of a pharmaceutical composition containing cells into a dysfunctional organ tissue and / or surrounding area. This improves conditions where lymphangiogenesis is impaired and improves disorders of tissue fluid homeostasis. For example, it may be a method of reducing edema, improving disorders in the ability to transport lipids and / or vitamins, , activating the immune surveillance mechanism, or treating organ failure.

[0048] In another embodiment of the first invention, the above-mentioned ADM and / or HHEX+ cells can be used as an injection or a transplant material for administration or transplantation to a failing organ tissue and / or its surrounding area, or a fibrotic tissue and / or its surrounding area. .

[0049] <Fibroblasts with high ADM expression> One embodiment of the second invention is fibroblasts with high ADM expression. The fibroblasts of this embodiment are preferably CD106 positive. They may also have high VEGF-C expression.

[0050] There is no limitation on the origin of the fibroblasts, and they may be differentiated from pluripotent stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and Muse cells, or adult stem cells such as mesenchymal stem cells. Additionally, primary cells collected from animals (including humans) may be used, or established cell lines may also be used. Examples of the organ from which the fibroblasts are derived include those from the lung, heart, kidney, skin, etc. The fibroblasts are not particularly limited, but are preferably of adult origin. The fibroblasts from which the cells of this embodiment are derived may originally express a certain amount of VEGF-C or ADM, or may not express it at all. In this embodiment, the fibroblasts may be cells that express cell surface markers expressed by normal fibroblasts, and further, the method for producing the cells of the present invention described later According to the law, in cells where additional markers are expressed or where some markers are not expressed It's okay to have it.

[0051] High expression of VEGF-C is not particularly limited, but for example, the mRNA expression level is R When measured using T-qPCR, if the mRNA expression level of control fibroblasts is set to 1, In the cells of this embodiment, the mRNA expression level of VEGF-C is 1.20 times or more, and 1.40 times or more. 1.50 times or more, 1.60 times or more, 1.70 times or more, 1.80 times or more, 1.90 2.00 times or more, 2.60 times or more, 2.99 times or more, 3.00 times or more, 3.38 3.52 times or more, 4.00 times or more, 4.11 times or more, 4.70 times or more, 5.00 More than 6.00 times, 7.00 times or more, 8.00 times or more, 9.00 times or more, 10.0 It is preferable that the increase is more than double, or by a multiple of more than that. Note that the mRNA expression level of control fibroblasts refers to the mRNA expression level of untreated fibroblasts. This could be the NA expression level, or the average mRNA expression level of the entire cell body after treatment. . Furthermore, when protein expression levels were measured using flow cytometry, VEGF-C The percentage of VEGF-C+ fibroblasts is 0 based on cell number. It may be 0.03% or more, 0.1% or more, 1% or more, and 2% or less It may be above, it may be 4% or more, it may be 5% or more, it may be 10% or more. It may be 20% or more, 30% or more, 40% or more, 50 It may be % or more, 60% or more, 70% or more, 80% or more It's fine if it's 90% or more, fine if it's 95% or more, fine if it's 98% or more. It can be 99% or higher.

[0052] High ADM expression is not particularly limited, but for example, it can be defined as reducing mRNA expression by RT-q When measured by PCR, if the mRNA expression level of control fibroblasts is set to 1, this procedure The mRNA expression level of ADM in morphological cells was 1.20 times or more, 1.40 times or more, 1. 43 times or more, 1.50 times or more, 1.60 times or more, 1.70 times or more, 1.80 times or more, 1. 90 times or more, 1.91 times or more, 1.97 times or more, 2.00 times or more, 2.46 times or more, 3. 00 times or more, 3.15 times or more, 4.00 times or more, 5.00 times or more, 6.00 times or more, 7. Multiples of 0.00, 8.00, 9.00, 10.0, or higher. It is preferable that it is added. Note that the mRNA expression level of control fibroblasts refers to the mRNA expression level of untreated fibroblasts. This could be the NA expression level, or the average mRNA expression level of the entire cell body after treatment. . Furthermore, when protein expression levels were measured using flow cytometry, ADM positivity The proportion of fibroblasts (hereinafter also referred to as ADM+) is 0.1% or more based on the cell number. It may be present, it may be 1% or more, it may be 2% or more, it may be 4% or more, 5 It may be % or more, 10% or more, 20% or more, 30% or more It's fine if it's 40% or more, fine if it's 50% or more, fine if it's 60% or more. It may be 70% or more, 80% or more, 90% or more, 95 It may be % or more, 98% or more, or 99% or more.

[0053] In this embodiment, the base sequences and amino acid sequences of VEGF-C and ADM are particularly While not limited to these, for example, VEGF-C is a base sequence represented by sequence number 13 or 14. A gene having, or a base sequence identical to that gene by 90% or more, 95% or more, or 98% or more. A gene having a base sequence, the entire base sequence represented by SEQ ID NO: 13 or 14 or It hybridizes with complementary sequences under stringent conditions, and VEGF -It may also be a gene encoding a protein having C activity, and ADM is sequence number A gene having a base sequence represented by 1 or 15, or a base sequence that is 90% or more, 95% Genes having the same base sequence as above or 98% or more, as expressed in Sequence ID No. 1 or 15 A complementary sequence to the whole or a part of the base sequence and hybridized under stringent conditions It may also be a gene that encodes a protein that is derived from soybeans and has ADM activity.

[0054] The cells of this embodiment preferably express CD106 as a cell surface marker.

[0055] This embodiment may also include a population of fibroblasts that express high levels of ADM as described above. stomach.

[0056] (Preparation of fibroblasts) In the preparation of fibroblasts, the origin of the fibroblasts is not particularly limited, as already mentioned. On the other hand, the cells may be of the patient's own origin, for example, lung tissue from a patient suffering from a lung disease. Lung fibroblasts isolated from patients, and lung fibroblasts isolated by differentiating adult (somatic) stem cells from patients. Cells may be prepared. Alternatively, pluripotent stem cells such as iPS cells derived from one's own cells may be differentiated. Furthermore, the collected fibroblasts may also be used. In addition, the cells may be other than the self, for example. Lung tissue isolated from donor-derived lung cells, or lung tissue produced using animals, etc. Prepare lung fibroblasts and lung fibroblasts isolated by differentiating adult (somatic) stem cells from a donor. It is also permissible to differentiate pluripotent stem cells such as donor-derived iPS cells or ES cells. The collected fibroblasts are shown in the picture.

[0057] (Isolation of fibroblasts) Prepared fibroblasts are typically supplied by enzymes such as dispase and collagenase. It can be separated by processing. Separation is done by enzymes such as dispase and collagenase. In addition to being carried out, if necessary separation is possible, other processes, such as mechanical processes, may also be performed. That's fine.

[0058] (Culture of fibroblasts) Fibroblasts may be brought into contact with TNF-α and IL-4 by the method described below, and desired fine cells may be produced. They may be subjected to culture until they reach a certain number of cells and / or until they possess the desired function. There are no restrictions on the culture conditions, and it may be carried out using known cell culture methods. The culture medium used for culturing can be appropriately set depending on the type of cells being cultured, for example, HFDM-1(+), DMEM, α-MEM, RPMI-1640, etc. are available. The culture medium contains nutrients such as NBCS, FCS, and FBS, as well as growth factors, cytokines, and antibiotics. Other substances may be added. Furthermore, the culture may be performed in a medium containing TNF-α and IL-4. The culture period is until the cultured cells reach the desired number of cells and / or until the cultured cells reach the desired number of cells. The culture time and number of culture days can be set appropriately depending on the purpose, such as until the function is acquired. For example, 1 Hours: 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 21 hours, 1 day, 2 days, 3 days, 4 days, 5 days, Periods such as 6 days, 7 days, 8 days, 9 days, 10 days, 2 weeks, 1 month, 2 months, 3 months, 6 months, etc. I can give it to you. The culture temperature can be set appropriately according to the type of cells being cultured, but for example, above 10°C. , may be 15℃ or higher, 20℃ or higher, 25℃ or higher, 30℃ or higher, and also 60℃ or lower, 5 The temperature may be 5°C or lower, 50°C or lower, 45°C or lower, or 40°C or lower. The culture process may be repeated multiple times. For example, the purity of the desired fibroblasts can be improved by selection or concentration. This allows for cultivation to be performed each time.

[0059] (Fibroblast harvesting) Cultured fibroblasts are harvested by detaching them using proteases such as trypsin. You may do so, but you can also use a temperature-responsive culture dish to detach the cells due to temperature changes and collect them. Often, cells can be detached and collected using instruments such as cell scrapers.

[0060] Another embodiment of the second invention is a method for producing ADM-highly expressing fibroblasts, wherein fibroblasts The process involves contacting cells with TNF-α and IL-4, and the fibroblasts that have been contacted with TNF-α and IL-4. This includes culturing fibroblasts that express high levels of ADM under conditions that can lead to high ADM expression. This is the manufacturing method. The fibroblasts of this embodiment are preferably CD106 positive. High expression of VEGF-C is also acceptable.

[0061] The method of contact between fibroblasts and TNF-α and IL-4 involves inducing fibroblasts that highly express ADM. The lead, or a line that is highly expressive of ADM and highly expressive of VEGF-C and / or positive for CD106. It is not particularly limited as long as fibroblast induction is not significantly impaired, and typically includes cultures containing fibroblasts. This involves adding TNF-α and IL-4 to the soil, but is not limited to this. IL-4 may be brought into contact with fibroblasts separately or simultaneously. For example, TNF-α and IL-4 can be mixed with water, culture medium, serum, or dimethyl sulfoxide (D It may be added to the culture medium after being dissolved in MSO.

[0062] In this embodiment, fibroblasts can be cultured in a medium supplemented with TNF-α and IL-4. Therefore, while maintaining the expression level of ADM in fibroblasts, furthermore, VEGF-C Fibroblasts can be cultured while maintaining the expression level of ADM. Furthermore, it is possible to produce fibroblasts with high VEGF-C expression levels. .

[0063] When adding TNF-α and IL-4 to the culture medium (mL), the amount of TNF-α added is not particularly limited. Although not fixed, it is usually 0.1 ng / mL or higher, and may be 0.5 ng / mL or higher. It may be 1 ng / mL or more, 10 ng / mL or more, and 50 ng / mL or more. It may also be the case. There is no particular upper limit, but it is usually 500 ng / mL or less, and 100 ng It may be less than / mL. Furthermore, while there are no particular restrictions on the amount of IL-4 added, it is usually 0.1 ng / mL or more. It may be 0.5 ng / mL or higher, it may be 1 ng / mL or higher, and it may be 2 ng / mL or higher. It is acceptable. There is no particular upper limit, but it is usually 10 ng / mL or less, and 5 ng / mL or less. It may be 2 ng / mL or less. The ratio (by weight) of TNF-α to IL-4 to be added is typically 100:1. It can be 00:1~1:1, or 50000:1~10:1. Also, 1:1~1 It is 10000, and can be anywhere from 1:10 to 1:50000.

[0064] Further culturing of fibroblasts contacted with TNF-α and IL-4 leads to high ADM expression. Fibroblasts can be produced. These fibroblasts are further characterized by high VEGF-C expression. That's fine. Fibroblast culture is not particularly limited as long as the conditions are such that fibroblasts can express high levels of ADM. It can be carried out using known cell culture methods. Also, conditions that can lead to high VEGF-C expression. The following conditions may also be met. It is preferable that the conditions are such that CD106 positivity can be achieved. The above fibroblast The method described in the section on (culture of fibroblasts) in the cell manufacturing method is an example.

[0065] In this embodiment, if fibroblasts are CD106 positive, an anti-CD106 antibody is used to A CD106-positive fibroblasts with high DM expression can be selected or enriched. It may also have high VEGF-C expression. The method of selection or concentration is not particularly limited, This can be done using methods known to the vendor, for example, human CD106(VCAM-1)-bioti Primary immunohistochemistry was performed using Miltenyi Biotec, and antibiotin microbiota Secondary immunostaining was performed using Miltenyi Biotec, and the stained cells were then auto-stained. This can be done by recovery at ACS (Miltenyi Biotec). Furthermore, selection or concentration can be performed at any time. Anti-CD106 antibodies can be any known type; commercially available products can be used. can. Selection or enrichment using an anti-CD106 antibody can lead to elevated ADM levels in fibroblast populations. The proportion of fibroblasts with increased expression can be increased.

[0066] The recovery of the cultured fibroblasts is exemplified by the method described in the section (recovery of fibroblasts ) in the above method for producing fibroblasts.

[0067] By the method for producing fibroblasts with high ADM expression of the present embodiment, fibroblasts with high ADM expression are obtained, and another embodiment of the second invention is a fibroblast population containing the fibroblasts or the cells. The fibroblasts may further have high VEGF-C expression and may be CD106 positive. When fibroblasts are stimulated by TNF-α and IL-4, the expression levels of ADM and V EGF-C increase. Therefore, fibroblasts with high ADM expression and further with high VEGF-C expression are preferably used as a therapeutic composition for diseases with reduced lymphangiogenesis ability, lung diseases such as pulmonary fibrosis or interstitial pneumonia, etc. In the present embodiment, the origin of the fibroblasts is not particularly limited, but is preferably derived from the lung. As another embodiment, it may be a fibroblast population containing cells obtained from the above-mentioned fibroblasts with high ADM expression.

[0068] <Cell population containing CD106-positive lung-derived fibroblasts> Another embodiment of the second invention is a cell population containing CD106-positive lung-derived fibroblasts. The fibroblasts of the present embodiment may have high ADM expression and may have high VEGF-C expression.

[0069] There is no restriction on the origin of the fibroblasts, and pluripotent stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and Muse cells, and adult stem cells such as mesenchymal stem cells are differentiated. It may also be used. Alternatively, primary cells collected from animals (including humans) may be used. Alternatively, established cell lines may be used. Fibroblasts are not particularly limited, but are preferably derived from adults. The fibroblasts from which the cells of this embodiment are derived originally contain a certain amount of VEGF-C and ADM. It may or may not be expressed at all. Furthermore, in this embodiment, fibroblasts refer to the cell surface on which normal fibroblasts express themselves. This may also refer to cells expressing a marker, and further, the method for producing the cells of the present invention described later. According to the law, in cells where additional markers are expressed or where some markers are not expressed It's okay to have it.

[0070] In a fibroblast population, the proportion of CD106-positive fibroblasts to the total number of fibroblasts is While not particularly limited, it is acceptable for the cell count to be greater than 18.01%, and greater than 27.97%. It's often over 30%, often over 31.38%, and often over 34.79%. It is fine if it is over 40%, fine if it is over 50%, fine if it is over 60%, fine if it is 70% It's fine if it's more than 80%, fine if it's more than 90%, fine if it's more than 95% It may be over 97.10%. Also, it may be 18.01% or more based on cell count. It may be 27.97% or higher, 30% or higher, or 31.38% or higher. It may be 34.79% or more, 40% or more, or 50% or more , may be 60% or more, may be 70% or more, may be 80% or more, and 90% It may be greater than or equal to 95%, or greater than or equal to 97.10%.

[0071] The fibroblast population of this embodiment is further a CD90-positive fibroblast population. At this time, in the fibroblast population, all fibroblasts were CD106-positive and CD9 The percentage of 0-positive fibroblasts is not particularly limited, but based on cell count, it was greater than 17.97%. It is fine if it is more than 27.91%, it is fine if it is more than 30%, and it is fine if it is more than 31.21% Often, it is often over 34.51%, often over 40%, often over 50%, and 6 It may be greater than 0%, may be greater than 70%, may be greater than 80%, may be greater than 90% It is good if it is over 95%, and good if it is over 96.29%. Also, based on cell count, 17 It may be 0.97% or higher, 27.91% or higher, and 30% or higher. It may be 31.21% or more, 34.51% or more, or 40% or more. It may be 50% or more, 60% or more, 70% or more, 80 It may be % or more, it may be 90% or more, it may be 95% or more, and 96.29% That's all.

[0072] Regarding the high expression of ADM and VEGF-C, see the above. <ADM The description in the section on "Highly Expressed Fibroblasts" can be used as a reference.

[0073] (Preparation of fibroblasts) In the preparation of fibroblasts, the origin of the fibroblasts is not particularly limited, as already mentioned. On the other hand, the cells may be of the patient's own origin, for example, lung tissue from a patient suffering from a lung disease. Lung fibroblasts isolated from patients, and lung fibroblasts isolated by differentiating adult (somatic) stem cells from patients. Cells may be prepared. Alternatively, pluripotent stem cells such as iPS cells derived from one's own cells may be differentiated. It may be the recovered fibroblasts. In addition, it may be cells other than oneself, for example Lung tissue derived from a donor providing lung cells, or isolated from lung tissue prepared using animals or the like Lung fibroblasts, or lung fibroblasts isolated by differentiating adult (somatic) stem cells of the donor may be prepared Moreover, pluripotent stem cells such as donor-derived iPS cells and ES cells may be differentiated to obtain the recovered fibroblasts.

[0074] (Isolation of fibroblasts) The prepared fibroblasts can typically be separated by treatment with enzymes such as dispase and collagenase. Separation can be carried out by treatment with enzymes such as dispase and collagenase. In addition to being performed by enzymes such as dispase and collagenase, other treatments, for example mechanical treatment, may be used if the required separation is possible.

[0075] (Culture of fibroblasts) The fibroblasts may be brought into contact with TNF-α and IL-4 by the method described later, and may be cultured for the purpose of achieving a desired number of cells and / or until they acquire a desired function. There is no limitation on the culture conditions, and it may be carried out by known cell culture methods. The medium used for culture can be appropriately set according to the type of cells to be cultured, etc. For example HFDM-1(+), DMEM, α-MEM, RPMI-1640, etc. can be used. Nutrients such as NBCS, FCS, and FBS, growth factors, cytokines, antibiotics, etc. may be added to the medium. Furthermore, it may be cultured in a medium containing TNF-α and IL-4. The culture period can be appropriately set according to the purpose, such as until the cultured cells reach a desired number of cells and / or until the cultured cells acquire a desired function. For example, 1 Hours: 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, 21 hours, 1 day, 2 days, 3 days, 4 days, 5 days, Periods such as 6 days, 7 days, 8 days, 9 days, 10 days, 2 weeks, 1 month, 2 months, 3 months, 6 months, etc. I can give it to you. The culture temperature can be set appropriately according to the type of cells being cultured, but for example, above 10°C. , may be 15℃ or higher, 20℃ or higher, 25℃ or higher, 30℃ or higher, and also 60℃ or lower, 5 The temperature may be 5°C or lower, 50°C or lower, 45°C or lower, or 40°C or lower. The culture process may be repeated multiple times. For example, the purity of the desired fibroblasts can be improved by selection or concentration. This allows for cultivation to be performed each time.

[0076] (Fibroblast harvesting) Cultured fibroblasts are harvested by detaching them using proteases such as trypsin. You may do so, but you can also use a temperature-responsive culture dish to detach the cells due to temperature changes and collect them. Often, cells can be detached and collected using instruments such as cell scrapers.

[0077] (Cell sorting of CD106-positive fibroblasts) If fibroblasts are CD106 positive, the selection of CD106-positive fibroblasts They may be subjected to selection or concentration. CD106-positive fibroblasts can be obtained without selection or concentration. If possible, selection or concentration may be omitted. Selection or concentration of CD106-positive fibroblasts This involves flow cytometry using anti-CD106 antibody (anti-VCAM-1 antibody), and magnetic biopsy. Examples of methods include the magneto-based method, affinity column method, or panning method. Specifically, Methods such as pneumatosis cell separation (MACS) and fluorescence-labeled cell separation (FACS) can be used. . Anti-CD106 antibodies can be readily commercially available or prepared by known methods and used. Either monoclonal antibodies or polyclonal antibodies may be used. However, from the perspective of specificity, it is preferable to use monoclonal antibodies. Further, by introducing a drug resistance gene and excluding CD106-negative fibroblasts, CD106-positive fibroblasts may be obtained. Also, by introducing a fluorescent protein-encoding gene and isolating fluorescent protein-positive cells using FACS or the like. Further, methods such as real-time PCR, next-generation sequencing and in situ hybridization may be used to confirm the expression of the CD106 gene in cells and isolate the CD106 gene expression group. <ADM highly-expressing fibroblasts or a cell population thereof, or a pharmaceutical composition containing a cell population of CD106-positive lung-derived fibroblasts>

[0078] Another embodiment of the second invention is a pharmaceutical composition containing the above ADM highly-expressing fibroblasts or a cell population thereof, or a pharmaceutical composition containing a cell population of the above CD106-positive lung-derived fibroblasts. The fibroblasts in this embodiment are preferably CD106-positive. Further, they may be highly-expressing VEGF- C. Since ADM and VEGF-C are lymphangiogenic factors, for example, by administering or transplanting the pharmaceutical composition of this embodiment to an organ such as the lung of a living body or its peripheral region, the state where the lymphangiogenic ability in an organ such as the lung is reduced can be improved. Therefore, although not particularly limited, for example, in an organ such as the lung, the state where the lymphangiogenic ability is reduced can be improved. It is particularly useful for the treatment of lung diseases such as pulmonary fibrosis or interstitial pneumonia. ADM and VEGF-C are lymphangiogenic factors. Therefore, for example, by administering or transplanting the pharmaceutical composition of this embodiment to an organ such as the lung of a living body or its peripheral region, the state where the lymphangiogenic ability in an organ such as the lung is reduced can be improved. Specifically, for example, in an organ such as the lung, the state where the lymphangiogenic ability is reduced can be improved. It is particularly useful for the treatment of lung diseases such as pulmonary fibrosis or interstitial pneumonia. Specifically, for example, in an organ such as the lung, the state where the lymphangiogenic ability is reduced can be improved. It is particularly useful for the treatment of lung diseases such as pulmonary fibrosis or interstitial pneumonia. . In this embodiment, the origin of the fibroblasts is not particularly limited, but is preferably derived from the lung. .

[0079] The method of administering or transplanting the pharmaceutical composition of this embodiment to a living body is not particularly limited. For example, it can be administered by injection. By directly administering or transplanting the pharmaceutical composition to the damaged area, fibrotic area or the surrounding area in organs such as the lung, improvement of the above disease state and treatment of organ diseases such as the lung can be achieved. In addition, the pharmaceutical composition may be administered to veins, arteries, lymph nodes, lymphatic vessels, bone marrow, subcutaneous tissue, corpus cavernosum, intraperitoneal cavity, muscle, intramedullary cavity, joint cavity. Administration to veins, arteries, and lymphatic vessels may be by injection into the blood vessel, may be by infusion through a catheter, or other known methods may be used. . The method of injection is not particularly limited, and known injection methods such as needle injection and needle-free injection can be applied. The method of the catheter used for infusion can also apply known methods and is not particularly limited. .

[0080] In addition, the pharmaceutical composition of this embodiment is not particularly limited, but may be applied to a living body as an artificial organ material. For example, the artificial organ material may be one in which cells are constructed as a planar or three-dimensional cell tissue. This planar or three-dimensional cell tissue may be one obtained by culturing fibroblasts alone, or may be a planar or three-dimensional cell tissue obtained by co-culturing fibroblasts with other cells, such as lung cells. Examples of the planar or three-dimensional cell tissue include, but are not particularly limited to, for example, cell sheets, cell fibers, and tissues formed by a 3D printer. . . In this embodiment, the size and shape of the planar or three-dimensional cell tissue depend on the damage or fibrosis. Applicable to the affected lung tissue and / or its surrounding areas and / or lesion areas, insofar as it is applicable, and not particularly limited. Furthermore, "planar" can refer to a single layer or multiple layers. Furthermore, conditions such as the culture medium composition, temperature, and humidity in cell culture are determined by VEGF-C and / or This allows for the culture of CD106-positive fibroblasts with high ADM expression, and in a planar or three-dimensional manner. The method is not particularly limited as long as it can construct a cell tissue in the form described above. For example, in the method for producing fibroblasts described above The culture conditions described in the section on (culturing fibroblasts) and / or (recovering fibroblasts) Similar examples can be given. Such planar or three-dimensional cell tissues, damaged or fibrotic organ tissues and / or so By applying it to the surrounding area, or as an artificial organ, damaged or fibrotic organ tissue and / Alternatively, by replacing it with surrounding tissue, the condition in which lymphatic vessel formation is reduced can be improved. It can also treat lung diseases such as pulmonary fibrosis or interstitial pneumonia.

[0081] The proportion of ADM-highly expressing fibroblasts contained in the pharmaceutical composition depends on the state of administration or transplantation to the patient. The formula is appropriately determined based on various factors, and the active ingredient is a therapeutically effective amount of fibroblasts that highly express ADM. It's fine as long as it's included. If the pharmaceutical composition contains other fibroblasts, for example, fibroblasts contained in the pharmaceutical composition The proportion of fibroblasts with high ADM expression relative to the total amount should be 0.03% or higher based on cell number. It may be 0.1% or more, 1% or more, 2% or more, and 4% or less It may be above, it may be 5% or more, it may be 10% or more, it may be 20% or more Often, 30% or more is fine, 40% or more is fine, 50% or more is fine, 6 It may be 0% or more, 70% or more, 80% or more, or 90% or more It may be, may be 95% or more, may be 98% or more, may be 99% or more, and is good.

[0082] The proportion of CD106-positive lung-derived fibroblasts contained in the pharmaceutical composition is appropriately set based on the mode of administration or transplantation, etc. As long as it contains a therapeutically effective amount of ADM-high-expressing fibro blasts as an active ingredient. Specifically, the proportion of CD106-positive lung-derived fibroblasts described in the above <Cell population containing CD106-positive lung-derived fibroblasts> can be cited. .

[0083] In addition, the number of ADM-high-expressing fibroblasts or the number of CD106-positive lung-derived fibroblasts contained in the pharmaceutical composition is, for example, 1.0×10 2 cells / mL or more, 1.0×10 3 cell s / mL or more, 1.0×10 4 cells / mL or more, 1.0×10 5 cells / mL or more, 1.0×10 6 cells / mL or more, 5.0×10 6 cells / mL or more, 1 .0×10 7 cells / mL or more can be used. The number of such cells contained in the injectable composition may be further increased or decreased according to the degree of the disease.

[0084] The method for confirming the above-mentioned therapeutic and / or improvement effects by the pharmaceutical composition is not particularly limited, For example, lymphangiogenesis increases after administration or transplantation of the composition compared to before administration or transplantation of the composition, the functionality of organs such as the lungs after administration or transplantation of the composition recovers compared to before administration or transplantation of the composition, and the degree of damage and fibrosis of organs such as the lungs after administration or transplantation of the composition is reduced compared to before administration or transplantation of the composition, etc. are exemplified.

[0085] The pharmaceutical composition may contain other components that are physiologically acceptable as a pharmaceutical composition. Other components include physiological saline, cell preservation solution, cell culture medium, hydrogel, and cells. Examples include outer matrix and cryopreservation solution.

[0086] Another embodiment of the second invention is the above-mentioned ADM-highly expressing fibroblasts, a cell population thereof, A cell population of CD106-positive lung-derived fibroblasts, or a pharmaceutical composition containing these, is used in the lungs. By administering or transplanting to any organ tissue and / or surrounding area, pulmonary fibrosis or interstitial fibrosis may occur. This may be a method for treating lung diseases such as pulmonary pneumonia. The fibroblasts in this embodiment are preferably It is CD106 positive. Furthermore, it may also have high VEGF-C expression. Furthermore, another embodiment of the second invention is the fibroblasts that highly express ADM, and the cells. A population, a cell population of CD106-positive lung-derived fibroblasts, or a pharmaceutical composition containing these. By administering or transplanting it into organ tissues such as the lungs and / or surrounding areas, lymphatic vessel regeneration occurs. This may be a method to improve a condition in which the bioactivity is reduced. The fibroblasts of this embodiment are preferred It is also CD106 positive. Furthermore, it may have high VEGF-C expression.

[0087] Furthermore, another embodiment of the second invention is the fibroblasts that highly express ADM, and the cells. Administration or transfer of a group or a composition containing them to organ tissues such as the lungs and / or surrounding areas. It may be used as an injectable agent or transplant material for implantation. The fibroblasts of this embodiment are Preferably, it is CD106 positive. Furthermore, it may also have high VEGF-C expression. [Examples]

[0088] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to the examples. Needless to say, it won't happen.

[0089] <Example A: A pharmaceutical composition containing fibroblasts with high expression of ADM and / or HHEX> Examples> <Increased CD106-positive cell rate in human adult-derived cardiac fibroblasts> We purchased an adult human heart from Science Care and administered Liberase MNP-S Human adult cardiac fibroblasts were isolated after enzymatic treatment with (Roche). 1% newborn calf serum (NBCS, Hyclo Recombinant human epidermal growth factor (rh-EGF)-containing fibroblasts with added ne Serum-free culture medium (HFDM-1(+), Cell Science & Technol The cells were cultured in ogy. After 5 subculturings, they were cultured in a cell culture dish, and the group cultured without treatment (N T), TNF-α (50ng / mL) (Peprotech) and IL-4 (2ng / m A group treated with a two-drug mixture of L)(Wako) (+CKs) was established. All groups were 1%N HFDM-1(+) with added BCS was used as the base medium, and the cells were incubated for 1 day (24 hours).

[0090] <Flow Cytometry> Fibroblasts are treated with BV421 mouse anti-human CD106 (VCAM1) antibody (BD Bi (osciences) and human CD90-PE (Miltenyi Biotec, Ber Immunofluorescence staining was performed using Gisch Gladbach (Germany). Isotype co The control is BV421 mouse IgG1;κ isotype control (BD Bi osciences) and REA control (S)-PE (Miltenyi Biot We used ec. The cells were reacted with the antibody while still alive for 30 minutes at 4°C. Immunofluorescence-stained cells are analyzed using a flow cytometer (MACSQuant Analyze). Analysis was performed using r 10 (Miltenyi Biotec). FSC-A and SSC-A were used. After recognizing the cell region, the percentage of cells positive for the CD106 protein (in percentage, converted to cell number) is evaluated. did.

[0091] <Increased ADM and / or HHEX gene expression in adult human cardiac fibroblasts> Human adult-derived cardiac fibroblasts were cultured in cell culture dishes, and the group cultured without treatment (NT) was selected. , TNF-α (50ng / mL) (Peprotech) and IL-4 (2ng / mL) A group treated with a two-drug mixture (Wako) (+CKs) was established. All groups received 1% NBC. HFDM-1(+) with added sulfur was used as the base medium, and the cells were incubated for 1 day (24 hours).

[0092] <Preparation of primers and probes> The primers used were human mRNA sequences (ADM,NM_) published by NCBI. 001124.3 (Sequence ID 1); HHEX, NM_002729.5 (Sequence ID 2); Based on ACTB(β-Actin), NM_001101.5 (Sequence ID 3), nearest neighbor The annealing temperature calculated by the base-pair method is around 60°C, and it contains one or more introns. The sequence was designed as follows (sequences 4-9). The 5' nuclease probe sequence is also publicly available. Designed based on human mRNA sequence data, the annealing temperature of this probe is determined by the primer. At temperatures more than 5°C higher, and where the binding site of this probe is located on the gene being measured, Designed to bind to the bases between the binding position of the word primer and the binding position of the reverse primer. In addition, the fluorescent dye 6-FAM was attached to the 5'-end of the probe, and the quencher dye ZEN was attached 9 bases away from there towards the 3'- side, and the second quencher dye Iowa Black FQ was attached to the 3'-end, making it a dual quencher probe that reduces background signals in RT-PCR analysis (SEQ ID NOs: 10-12). All of the above primers and probes were commissioned for synthesis by Integrated DNA Technologies Inc. (Table 1). (Table 1)

Table 1

[0093] <RNA Extraction> Total RNA was extracted from cardiac fibroblasts using the Qiagen RNeasy Plus M ini Kit (QIAGEN). At that time, cell lysates were prepared using QI Ashredder (QIAGEN), and other operations including DNase treatment were performed according to the recommended protocol of the kit manufacturer. The concentration of the obtained total RNA was measured using a N anodrop One / One spectrophotometer (Thermo Fisher Scien tific) to measure the absorbance at a wavelength of 260 nm. At the same time, the value of A260 / A280 indicating the contamination level of proteins was calculated, and it was confirmed that A260 / A280 > 2.0 . was confirmed.

[0094] <Real-Time Quantitative RT-PCR> One Step PrimeScript 3 RT-qPCR Mix, with UNG Kit (TaKaRa) was used to perform reverse transcription reaction and RT-PCR in the same tube​ I did it all together. The response was 1 x One Step PrimeScript 3 RT -qPCR Mix, with UNG, 0.2μM Forward & Rever Reaction solution containing se primer, 0.2 μM specific probe, and 10 ng template RNA solution. The procedure was carried out with a total volume of 12.5 μL. The reverse transcription reaction was first performed by heating at 52°C for 5 minutes. The process was then carried out, and subsequently heated at 95°C for 10 seconds to inactivate the reverse transcriptase. The cycle of heating at 60°C for 5 seconds and then heating for 30 seconds was repeated 40 times, and fluorescence was detected for each cycle. The intensity was measured. The series of reactions and measurements were performed using a Thermal Cycler Dice Re The analysis was performed using the al Time System 3 (TaKaRa). Simultaneously, a calibration curve was created. RNA extracted from samples in which the expression of ADM and HHEX has been confirmed was used as a reference for the analysis. Using a solution, the above measurements were performed using RNA solutions diluted 10-fold in four steps, starting from 1 μg. All samples were measured with N=3. After PCR is complete, the obtained amplification curves are analyzed, and the baseline fluorescence intensity for each target gene is measured. A threshold is set, and the number of cycles (Ct value) in which the fluorescence signal reaches that threshold is recorded for each sample. These analyses were performed using Thermal Cycler Dice Real Timing. This was done using e System 3 Software (TaKaRa). A gene calibration curve has a correlation coefficient of 0.98 or higher and an amplification efficiency of 90% or higher, and is considered a calibration curve. Since it was considered sufficient, the Ct values ​​of the target samples were fitted to the calibration curve and the m of the target gene was determined. RNA content was calculated as a relative value. This relative value was then standardized using the β-Actin value.

[0095] <Lymphatic vessel vascularization assay> Lymphatic vessel vascularization assays are performed by methods known to those skilled in the art, using cardiac-derived cells. Human lymphatic endothelial cells and various human cardiac fibroblasts were co-cultured, and various cardiac fibroblasts The effect of cardiac lymphangiogenesis was evaluated (Non-Patent Literature 15). Specifically, human heart Human lymphatic endothelial cells and various types of adult human cardiac fibroblasts were collected from the derived cells. 45×10 5 cells / cm 2 Seeds were sown at a concentration of 2:8, and EGM-2MV Mi crovascular Endothelial Cell Growth Medi With um-2 BulletKit (Lonza, Basel, Switzerland) The cells were co-cultured for 3 days. After co-culture, the cells were fixed with 4% paraformaldehyde and treated with 0.1% Tr After membrane permeabilization with itonX, immunofluorescence staining was performed. The primary antibody used was anti-VE-Cadher in rabbit polyclonal antibodies (abcam, Cambridge, UK) and anti-Vime The ntin mouse polyclonal antibody (abcam) was used. The secondary antibody was goat antirabbit Goat IgG H&L (Alexa Fluor 488) (abcam) and goat anti-mouse I gG H&L (Alexa Fluor 647) (abcam) was used for nuclear staining. The Hoechst 33258 solution (Dojin Chemical Research Institute, Kumamoto, Japan) was used. The stained samples were analyzed using the IN Cell Analyzer 2200 (Cytiva, M). This photo was taken in Arlborough, MA. Quantitative evaluation was performed using Analyzer for ImageJ (v. 1.0.c, Gill es Carpentier,2012;available online:http : / / imagej.nih.gov / ij / macros / toolsets / Ang I used iogenesis%20Analyzer.txt. Specifically, Icy (v.2.0.3.0;BioImage Analysis Lab,Institu Using t Pasteur, we extracted the channels of interest and subtracted the background. After that, an anisotropic PDE-based filter (Icy Plug Apply “Membrane Filter”, v.0.1.0.1) to correct the image. Then, enhance the contrast of the filtered image, normalize it, and subtract the background. The ROI was defined using Angiogenesis Analyz. Finally, the obtained images were analyzed using Angiogenesis Analyz. The data was analyzed using the er macro. For detailed protocol information, see dx.doi.org / 10.17504 It has been published online at / protocols.io.bhtaj6ie. The quantitative data illustrates the total length and number of junctions of mature lymphatic vessels, with the average NT value set to 1. This was then represented using Fold increase.

[0096] <Preparation of cells for transplantation> The cells were purchased from the following manufacturer: Human adult-derived cardiac fibroblasts (Lonza); Fetal-derived cardiac fibroblasts (Cell Applications, San Diego) ,CA). The cells are cultured in fibroblast culture medium (HFDM-1(+)medium (Cell Corporation). The cells were cultured on a large scale at the Institute of Scientific Research (Miyagi, Japan) and used in transplantation experiments.

[0097] <Cell sorting> Fibroblasts are human CD106(VCAM-1)-biotin (Miltenyi Bi (otec), or human CD90-biotin (Miltenyi Biotec) as primary Immunostaining was performed, and two types of antibiotin microbeads (Miltenyi Biotec) were detected. Next, immunostaining was performed. The stained cells were identified using autoMACS (Miltenyi Biotec). CD106 and CD90-positive cells were collected.

[0098] <Creation of a rat model for chronic heart failure> All animal experiment protocols used in this study were developed by LSI Medience (Tokyo, Japan). The ethics review committee approved the study. Furthermore, pain relief measures were implemented for all animals. Nude rat (F344 / N Jcl-rnu / rnu), 8 weeks old, male, from Japan CREA (East Purchased from Kyoto, Japan. After more than a week of acclimatization, the animals were administered using an inhalation anesthesia machine for laboratory animals (soft lava). Using Wander (Shin-ei Kogyo Co., Ltd., Saitama, Japan), 2% isoflurane (anesthetic adjuvant; lol) The animal was anesthetized by inhalation with oxygen (7:3 ratio) and its fur was shaved. Endotracheal intubation was performed quickly, and the animal was then 0.5 -2% isoflurane (anesthetic adjuvant; nitrous oxide:oxygen = 7:3) inhalation anesthetic gas is applied to the ventilator. Anesthesia was then maintained. The patient was fixed in a supine position under mechanical ventilation, with the left third to fifth ribs. Two to three longitudinal cuts were made at the location of the costal cartilage, leading to the thoracotomy. The surgical field was then opened using a retractor. After magnification, the pericardium was dissected to expose the heart. The left atrium was lifted, and a softly curved round needle with vascular sutures was used. Using (6-0: Nesco suture), thread the suture through the left ventricle at a depth of approximately 2 mm and a length of 4-5 mm. He did so. He joined the ends of the thread together and passed a snare made of polyethylene tubing through it, then used an arterial clamp. Using a snare, the coronary artery was ischemiaed for 30 minutes by tightening a suture. After 30 minutes, reperfusion was performed, and the patient's condition was assessed. Once the condition stabilized, we confirmed there was no bleeding, performed chest tube drainage, and sutured the muscle layer and skin. The skin is sutured intradermally, and if a standard suture is used, the sutures are removed while observing the postoperative condition. The following procedure was performed: One week after model creation, on the day before cell administration, an ultrasound imaging device (Xario) was used. Echocardiography was performed using the SSA-660A (Toshiba Medical Systems, Tochigi, Japan). The left ventricular ejection fraction (LVEF = (LVIDd3 - LVIDs3) / LVIDd3) was 55%. The following individuals were considered a model of chronic heart failure, and experiments involving the administration of fibroblasts were conducted.

[0099] <Administration of fibroblasts to a rat model of chronic heart failure> On the day of the administration test, various fibroblasts that had been cryopreserved were thawed and high-glucose Diluted with DMEM + 10% NBCS, the number of viable cells per individual was 2.0 × 10⁶. 6 The animals were administered a cell suspension of 50 μL cells / 50 μL. The animals were anesthetized using the same method as when the model was created. While maintaining intoxication and under mechanical ventilation, a catheter with a 30G needle was inserted into two separate locations within the infarcted area. The entire 50 μL of cell suspension was administered using [a specific method]. After the patient's condition stabilized, leakage of the administered solution and bleeding occurred. After confirming the absence of any abnormalities, chest drainage was performed, and the muscle layer and skin were sutured. The skin was sutured intradermally. However, if suturing was performed, the sutures were removed while observing the postoperative condition.

[0100] <Collection of heart tissue and tissue staining> Rats were sacrificially killed 18 weeks after cell administration, and their hearts were collected. The collected hearts were treated with 4% paraform. After fixation with mualdehyde, paraffin sections were prepared by paraffin embedding, and frozen sections were also prepared. The following was prepared: Paraffin sections were stained with Sirius Red (SR) and hematoxylin. The samples were stained with ¹H₂O₃ (HE) and used in situ hybridization. The group administered somatic cardiac fibroblasts used a virtual slide scanner (NanoZoomer S Images were taken using 210 (Hamamatsu Photonics, Shizuoka, Japan). The group administered with fetal-derived cardiac fibroblasts was A Images were captured using a perio ScanScope slide scanner. SR stained image. The images were taken using ImageJ for the group administered adult-derived cardiac fibroblasts, and for the group administered fetal-derived cardiac fibroblasts. The group receiving cytoplasmic respiration (HALO) (Indica Labs, Albuquerque, NM) showed signs of fibrosis. The colored region was quantified. HE-stained images were evaluated qualitatively using the following criteria: Infl ammatory cell aggregation, proliferation of fibroblasts, fibrosis, lime deposition, Thinning of the left ventricular. Degree of pathological changes. The scores are: no change (score 0), mild (score 1), moderate (score 2), and severe (score 3). It was qualitatively evaluated as follows: In situ hybridization was performed using RNAsc. ope Target Probe Rn-Lyve-1-C2(Bio-Techne Using Minneapolis (MN), staining was performed according to the manufacturer's recommended protocol. The probes were processed using a HybEZ Oven (Bio-Techne). . Frozen sections were treated with anti-cardiac troponin T (cTnT) antibody (Abcam , Cambridge, UK), anti-prospero-related homeobo x 1(Prox-1) antibody (Proteintech, Rosemont, IL), and and anti-vascular endothelial growth factor rec eptor 3 (VEGFR3) antibody (Bioss Antibodies, Wobur Immunohistochemical staining was performed using n, MA, and an FV1200 confocal laser microscope (Olympus) was used. Images were taken in Tokyo, Japan.

[0101] <Statistical analysis> The mean values ​​of the NT group and the +CKs group were compared using Student's t-test for statistical analysis. .

[0102] <Example A1: CD1 in adult-derived cardiac fibroblasts upon contact with TNF-α and IL-4> 06Improvement of positive cell rate> Human adult-derived cardiac fibroblasts were cultured in cell culture dishes, and two groups were established: an NT group and a +CKs group. All groups of human adult-derived cardiac fibroblasts were flattened and spindle-shaped, and had a typical linear structure. They had a fibrous cell-like shape (Figure 1A). Flow cytometry: Percentage of CD90, CD106, and both CD90 and CD106 positive cells ( When the percentages were evaluated, the NT group showed 48.88% (CD90) and 0.20% (C). D106) showed 0.14% (both positive, DP), while the +CKs group showed the following: The percentages were 57.55% (CD90), 50.78% (CD106), and 34.76% (DP). The percentage of CD90-positive cells, CD106-positive cells, and the percentage of cells positive for both (%) improved significantly. Figure 1B). These results suggest that by contacting fibroblasts with TNF-α and IL-4, The study showed an improvement in the percentage of fibroblasts that are positive for both CD90 and CD106.

[0103] <Example A2: Contact with TNF-α and IL-4 results in adult-derived cardiac fibroblasts developing odor The expression levels of ADM and / or HHEX were enhanced. Total RNA was extracted from adult-derived cardiac fibroblasts of all groups (NT group, +CKs group). After quality control of the RNA sample, a probe detection system (5' nuclease method) is used. The fluorescence intensity correlated with the mRNA copy number is detected, and the phase of mRNA copy number is determined using a calibration curve method. The logarithm was calculated (N=3). Gene expression of ADM and HHEX in fibroblasts of all groups. The amount was standardized by the gene expression level of β-Actin, and the ADM and / or HHEX of the NT group The gene expression level (average value) was set to 1.00 and expressed as a fold increase. The results showed that ADM expression levels increased 2.30-fold in the +CKs group, indicating significant mRN A difference in the expression level of A was confirmed (Figure 2A). On the other hand, the expression level of HHEX was 2 in the +CKs group. A 0.58-fold improvement was demonstrated, and significant differences in mRNA expression levels were confirmed among all groups. (Figure 2B). These results suggest that contacting fibroblasts with TNF-α and IL-4 is effective. This has been shown to improve the gene expression levels of ADM and / or HHEX in fibroblasts. It was done.

[0104] <Example A3: Adult-derived cardiac fibroblasts that came into contact with TNF-α and IL-4 developed cardiac phosphorus The effect of regenerating pharmacokinetics is high. To evaluate the lymphangiogenic effect of adult-derived cardiac fibroblasts treated with TNF-α and IL-4. Therefore, human cardiovascular endothelial cells containing lymphatic endothelial cells were cultured on a large scale. Using lymphatic endothelial cells that are positive for both VE-cadherin and PDPN, the test was conducted. An experiment was conducted. Human lymphatic endothelial cells and various adult human cardiac fibroblasts recovered from cardiac-derived cells. When these were co-cultured and lymphatic vessels were observed by immunofluorescence staining, untreated adult-derived cardiac wires were found to be different. Fibroblasts (NTs) exhibit discontinuous vascular formation where lymphatic vessels are fragmented, whereas TNF-α Adult-derived hearts were cultured for 24 hours after adding (50 ng / mL) and IL-4 (2 ng / mL). Visceral fibroblasts (+CKs) are known to form continuous, uninterrupted blood vessels. This was the result (Figure 3A). Furthermore, quantitative analysis also showed that +CKs significantly increased mature lymphatic vessel activity. The overall length was extended to 2.21 ± 0.76 times that of NT (Figure 3B). Number of junctions A significant increase was also observed, with +CKs increasing 1.84 ± 0.65 times compared to NT. Figure 3B). From these results, adult-derived cardiac fibroblasts treated with TNF-α and IL-4 showed phosphorus It was revealed that these cells have a high efficacy in vascular regeneration.

[0105] <Example A4: When TNF-α and IL-4 were brought into contact, both CD90 and CD106 tested positive.> Adult-derived cardiac fibroblasts have therapeutic effects on fibrosis. Lymphangiogenesis can treat scar formation (fibrosis) that occurs as a result of organ tissue damage. This has been reported in many previous studies. To confirm this effect, first we will examine the following adult-derived heart Fibroblasts were prepared (Figure 4): Untreated adult-derived cardiac fibroblasts (A-HCF);TN Adults cultured for 72 hours after being treated with F-α (50 ng / mL) and IL-4 (2 ng / mL). Derived cardiac fibroblasts (uA-HCF); among uA-HCF cells, those positive for both CD90 and CD106. The cell population was sorted using MACS with CD90 as the indicator, and the collected cells were... Group (uA90-HCF). Note that naturally occurring CD90 and CD106-positive cardiac fibers. For comparison with blast cells, fetal-derived cardiac fibroblasts were analyzed using MACS with CD106 as an indicator. Cell sorting was performed and the cells were collected (F-VCF). The characteristics of each fibroblast were evaluated by FCM. The results of the evaluation are shown in Figure 4. The following cells were administered to a rat model of chronic heart failure, and the fibrotic area was observed using Sirius Red staining. The following was performed (Figure 5). Study 1: A-HCF administration group; uA-HCF administration group; uA90-HCF Treatment group; Control group (group administered only culture medium); Sham group (group subjected to only sham surgery); Microscopic observations were performed in each group (the group that did not develop the failure) (Figures 5A and 5B). Experiment 2 Microscopic examination was performed on each group: the F-VCF administration group and the control group (the group that received only culture medium). Observations were made (Figures 5C and 5D). As a result, TNF-α (50 ng / mL) and IL-4 ( Fibroblasts treated with 2 ng / mL of TNF-α showed a smaller area of ​​myocardial fibrosis, particularly in TNF-α Only the cell population that became positive for both CD90 and CD106 due to the effect of IL-4 was administered. The fibrotic region was remarkably small in the group (uA90-HCF). Interestingly, it is naturally occurring. The results of administering CD90 and CD106-positive cardiac fibroblasts (F-VCF) to a group of patients were as follows: Therefore, no therapeutic effect was observed in the fibrotic area. Based on these results, TNF-α (50ng Lymphatic fibroblasts artificially created by adding ( / mL) and IL-4 (2ng / mL) We were able to identify functional differences in fibrosis of the cysts. Furthermore, we were able to identify differences in the functionality of various adult-derived cardiac fibers. Pathological evaluation based on HE-stained images of the group administered blast cells showed the same results as SR staining. Fibroblasts treated with TNF-α (50 ng / mL) and IL-4 (2 ng / mL) Proliferation of fibroblast, Fibrosis, Li The me deposition score is showing a decreasing trend, particularly regarding the effectiveness of TNF-α and IL-4. In the group that received only the cell population that became positive for both CD90 and CD106 as a result (uA90- In HCF, each of these values ​​decreased significantly (Figure 6). Next, in situ hybridization, lymphatic vessel intracellular markers were used. a lymphatic vessel endothelial receptor We stained the lymphatic vessels with 1 (Lyve-1) and observed the lymphatic endothelial cells recruited to the myocardial infarction lesion. As a result, similar to the results of SR staining and HE staining, TNF-α (50 ng / mL) and IL-4 ( Fibroblasts treated with 2 ng / mL showed a mobilization effect on lymphatic endothelial cells (lymphangiogenesis effect). The levels were high, and in particular, the effects of TNF-α and IL-4 resulted in positive results for both CD90 and CD106. The effect was significantly increased in the group administered only with the cell population (uA90-HCF) (Figure 7). ).

[0106] <Example A5: Lymphatic vessels in vivo by cardiac fibroblasts positive for both CD90 and CD106> Rejuvenation effect > In a rat model of chronic heart failure, fibroblasts (A-HCF, uA-HCF, or uA90- HCF) or Vehicle Control (culture medium only) is administered intracardiacally, and each Lymphangiogenesis in vivo was observed in the group (Figure 8). Immunohistochemical observations revealed that uA Only in the 90-HCF administration group were the lymphatic endothelial cell (LEC) markers Prox-1 and VEGFR3 localization was confirmed, but LEC localization was not confirmed in the other groups (Figure 8). ).

[0107] <Example B: Example concerning fibroblasts with high ADM expression> <Culturing of human lung fibroblasts and generation of fibroblasts with high ADM expression and enhanced VCAM-1 expression> > Human adult lung fibroblasts are PromoCell (Heidelberg, Germany) Purchased from ) and added 5% (v / v) NBCS (Newborn Calf Serum) Fibroblast culture medium (HFDM-1(+)medium (Cell Science Institute Co., Ltd.) Human pulmonary microvascular endothelial cells were cultured in large quantities at Lonza (Basel, S) in Miyagi, Japan. The cells were purchased from Witzerland and cultured in EBM-2 medium (Lonza). Visual field observation was performed using a Leica DMi1 (Leica GmbH, Wetzlar, Germ). I went with any) To generate fibroblasts with high ADM expression and enhanced VCAM-1 expression, human lung fibroblasts Cells were cultured in cell culture dishes, and one group was cultured without treatment (Control), while the other group was cultured with TNF- α (50ng / mL) (Peprotech, Cranbury, NJ) and IL-4 (2 The group treated with a mixture of two drugs (ng / mL) (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) +Factor-X) was added. All groups were HFDM-1 with 5% NBCS added. The culture medium (+) was used as the base medium, and the cells were incubated for 1 day (24 hours).

[0108] <Flow Cytometry> Human lung fibroblasts are human CD90-PE (Miltenyi Biotec, Ber Gisch Gladbach (Germany) and BV421 mouse anti-human CD1 Immunofluorescence staining was performed with 06 (BD Biosciences, San Jose, CA). The isotype control is REA control (S)-PE (Miltenyi B iotec) and BV421 mouse IgG1 κ isotype control (BD B (iosciences) was used. On the other hand, mouse lung fibroblasts were used in mouse CD90.2 -PE (Miltenyi Biotec) and mouse CD106 (VCAM-1)-A Immunofluorescence staining was performed using PC (Miltenyi Biotec). Isotype control The rat IgG2b-PE (Miltenyi Biotec) and rat IgG2 a-APC (Miltenyi Biotec) was used. The cells were kept alive and mixed with the antibody. The cells were reacted at 4°C for 0 minutes. Immunofluorescence-stained cells were then measured using a flow cytometer (MACSQ). Analysis was performed using uant Analyzer 10 (Miltenyi Biotec). After recognizing the cellular region with SC-A and SSC-A, the CD90 and CD106 proteins are targeted. The percentage of positive cells (in percentage, converted to cell number) was evaluated.

[0109] <Evaluation of gene expression levels in pulmonary fibroblasts> The primers used were human mRNA sequences (VEGF-C, VEGF-C) published by NCBI. NM_005429.5 (Sequence ID 13); ADM, NM_001124.3 (Sequence ID 1); ACTB (β-Actin), NM_001101.5 (Sequence ID 3)) and M Us mRNA sequence (VEGF-C,NM_009506.2(SEQ ID NO: 14);ADM, NM_009627.2 (SEQ ID NO: 15); GAPDH,NM_001289726.1 Based on (Sequence No. 16), the annealing temperature calculated by the nearest-neighbor base pair method is around 60°C. So, the sequence was designed to enclose one or more introns (sequences 17-18 (Human V) EGF-C primer), SEQ ID NOs. 4-5 (human ADM primer), SEQ ID NOs. 8-9 ( Human ACTB primers), SEQ ID NOs. 19-24 (mouse VEGF-C primers, mouse ADM primer, mouse GAPDH primer). 5' nuclease probe sequence. This probe was also designed based on publicly available human and mouse mRNA sequence data, and this probe The Neil temperature is more than 5°C higher than that of the primer, and the binding site of this probe is the target of measurement. The binding sites of the forward primer and the reverse primer on the gene. It was designed to bind to the bases in between. Additionally, the 5' end of the probe was treated with the fluorescent dye 6-FAM. 9 bases from there, 3', is the quencher dye ZEN, and at the 3' end is the second quencher dye Dual quencher probe conjugated with enchantment dye Iowa Black FQ This reduces background signal in RT-PCR analysis (SEQ ID NO: 25 (Human VEGF-C probe), SEQ ID NO: 10 (Human ADM probe), SEQ ID NO: 1 2 (human ACTB probe), SEQ ID NOs. 26-28 (mouse VEGF-C probe, mouse ADM probe, mouse GAPDH probe). All of the above primers and probes are The synthesis was outsourced to Integrated DNA Technologies (Table 2). . [Table 2] Total RNA extraction from lung fibroblasts is performed using Qiagen RNeasy Plus Mi The procedure was performed using the ni Kit (QIAGEN). During this process, cell lysates were disrupted using QIA. This is done using shredder (QIAGEN), and other operations including DNase processing are performed. The procedure followed the kit manufacturer's recommended protocol. The concentration of the obtained total RNA was measured using Na nodrop One / One Spectrophotometer (Thermo Fisher Scient The absorbance at a wavelength of 260 nm was measured using an ific (physical crystal). Simultaneously, the protein's Calculate the A260 / A280 value, which indicates the degree of contamination, and ensure that A260 / A280 > 2.0. I confirmed it. Real-time quantitative RT-PCR is performed using One Step PrimeScript 3. Reverse transcription using RT-qPCR Mix, with UNG Kit (TaKaRa) The reaction and RT-PCR were performed together in the same tube. The reaction was 1×One Step PrimeScript 3 RT-qPCR Mix, with UNG, 0.2μM Forward & reverse primers, 0.2 μM specific probe, 10 ng (human lung radiation) Reaction solution containing template RNA solution (total) fibroblasts (or 100 ng (mouse lung fibroblasts)) The procedure was performed using 15 μL of solution. First, the reverse transcription reaction was carried out by heating at 52°C for 5 minutes. Afterward, the reverse transcriptase was inactivated by heating at 95°C for 10 seconds. Then, it was heated at 95°C for 5 seconds. The process involved heating at 60°C for 30 seconds, repeated 40 times, and the fluorescence intensity was measured for each cycle. Determined. The series of reactions and measurements were performed using 7500 Rast Real-Time PCR (Th The procedure was performed using Fisher Scientific (ERMO). Simultaneously, a calibration curve was created. As a reference material, RN was extracted from a sample in which the expression of ADM and VEGF-C had been confirmed. Using solution A, the above measurements were performed using RNA solutions diluted 10-fold in four steps, starting from 1 μg. All samples were measured with N=3. After PCR is complete, the obtained amplification curves are analyzed, and the baseline fluorescence intensity for each target gene is measured. A threshold is set, and the number of cycles (Ct value) in which the fluorescence signal reaches that threshold is recorded for each sample. The calibration curve for each gene was determined to have a correlation coefficient of 0.99 or higher and an amplification efficiency of 85% or higher. Since it was considered sufficient as a calibration curve, the Ct values ​​of the target samples were applied to the calibration curve. The mRNA content of the target gene was calculated as a relative value. This relative value was then calculated in the same way. The values ​​were standardized using the housekeeping gene (β-Actin or GAPDH) that was extracted. Furthermore, the gene expression levels of fibroblasts were standardized using housekeeping gene expression levels. The gene expression level of trol was set to 1, and the result was expressed as a fold increase.

[0110] <Lymphatic vessel vascularization assay> Lymphatic vessel vascularization assays are performed using methods known to those skilled in the art, and are performed from human lung-derived cells. Lymphatic endothelial cells were co-cultured with various human lung fibroblasts, and lung lymphocytes were cultured using various lung fibroblasts. We evaluated the effect of pulmonary angiogenesis (Non-Patent Literature 15). Specifically, we used cells derived from human lung cells. Collected lymphatic endothelial cells and various adult human lung fibroblasts were divided into 2.5 × 10⁻⁶ cells. 5 cells / cm 2 Seeds were seeded at a concentration of 1:9 and co-cultured in EBM-2 medium for 3 days. After co-culture, Cells were fixed with 4% paraformaldehyde and immunofluorescence staining was performed. The primary antibody used was anti-VE. -Cadherin rabbit polyclonal antibody (abcam, Cambridge, UK) ) and anti-Vimentin mouse polyclonal antibody (abcam) were used. Secondary antibody This is goat anti-rabbit IgG H&L (Alexa Fluor 488) (abcam) and Goat anti-mouse IgG H&L (Alexa Fluor 647) (abcam) was used. Nuclear staining was performed using Hoechst 33258 solution (Dojin Chemical Research Institute, Kumamoto). The study was conducted in Japan. Stained samples were prepared using an IN Cell Analyzer 2200 (C This photo was taken in Ytiva, Marlborough, MA.

[0111] <Isolation and primary culture of mouse lung fibroblasts and fibroblasts with high ADM expression and enhanced VCAM-1 expression> Blast cell production > All animal experiments conducted in this study were performed at the Nano Medical Innovation Center (Kanagawa, Japan). It was approved by the Ethics Review Committee (Animal Experiment Protocol Approval Number: A20-003). Pain relief measures were implemented for all animals. Mouse C57BL / 6J (male, 10 weeks old) is from Charles River, Japan (Kanagawa, Japan) Purchased from ). Mouse adult-derived lung fibroblasts are primary cells harvested from lung tissue. These primary cells were then cultured on a larger scale. Specifically, the harvested adult cells were cultured on a larger scale. The lung tissue (C57BL / 6 mouse, 10 weeks old) was minced into 1 mm squares with a scalpel, and 0.14 Liberase TM (Sigma Aldri) in Wunsch units / mL (ch, St. Louis, MO) was added and the enzyme treatment was carried out in a 37°C incubator. Primary lung fibroblasts were isolated and cultured. The culture medium was supplemented with 15% FBS. The added DMEM / F12 medium was used. Bright-field observation was performed using a Leica DMi1 (Leic This was conducted at a GmbH (Wetzlar, Germany). To generate adult-derived lung fibroblasts (VPFs) with high ADM expression and enhanced VCAM-1 expression. Therefore, adult mouse lung fibroblasts were cultured in a cell culture dish, and the group cultured without treatment was... (Control) and recombinant mouse TNF-α (50 ng / mL) (Pepr (otech) and recombinant mouse IL-4 (2ng / mL) (Peprotech) A group treated with a mixture of two agents (+Factor-X) was established. All groups were treated with 15% FBS. Using DMEM / F12 medium supplemented with A as the base medium, the culture was performed for 3 days (72 hours). We created adult-derived lung fibroblasts (VPF) with high DM expression and enhanced VCAM-1 expression. The VPF described below was used in Examples B3 and B4. Furthermore, using a similar method, C5 was purchased from Charles River Japan (Kanagawa, Japan). Lungs were collected from 7BL / 6 mice (female, 10 weeks old) and primary cultures were performed to extract lung fibroblasts (P Expand culture of F) and further develop ADM-highly expressing and VCAM1-enhanced PF (VPF). It was fabricated. This VPF was used in Example B5 described later. In this specification and in the figures, mouse-derived PF is also referred to as mPF, and rat The PF derived from a mouse is sometimes called rPF. Also, the VPF derived from a mouse is sometimes called mVPF. In some cases, rat-derived VPF is called rVPF.

[0112] <Creation of mouse models of interstitial pneumonia and pulmonary fibrosis> Adult-derived lung with high ADM expression and enhanced VCAM-1 expression for interstitial pneumonia and pulmonary fibrosis To evaluate the therapeutic effect of fibroblasts (VPF), bleomycin-inducible mouse C57B I purchased the L / 6J model from Charles River Japan. (Bleomycin-induced interstitial lung) Induction of inflammation and pulmonary fibrosis was carried out by methods known to those skilled in the art (Non-Patent Literature 16). Specifically This involves using bleomycin solution (1 U / ml in 0.9% physiological saline) in liquid Mic Fill roSprayer (PennCentury, Wyndmoor, PA) and 5 The drug was administered via the airway to the lungs of 9-week-old male mice under deep anesthesia at a concentration of 2 mg / kg per 0 mL. Mice 7 days after administration of bleomycin solution were considered a model of interstitial pneumonia and pulmonary fibrosis.

[0113] Evaluation of the therapeutic effect of cell administration and post-administration VPF in mouse interstitial pneumonia and pulmonary fibrosis models. > Fibroblasts were administered intravenously via the tail vein to interstitial pneumonia / pulmonary fibrosis model mice. Fibroblasts are 1.0 × 10 5 Cells were administered in a 100 μL solution of physiological saline per body. Each mouse was given mPF, mVPF, or physiological saline (Ctr) as a control. (l) was administered. The therapeutic effect of each cell was determined by survival analysis using the Kaplan-Meier method and pathological evaluation. The experiment was conducted as follows: In Examples B3 and B4, the results were obtained 4 weeks (28 days) after cell administration, which was the end of the experiment. Animals were sacrificed and their lungs were collected. The collected lungs were fixed in 4% paraformaldehyde. Afterward, paraffin embedding was performed. The paraffin sections were stained with Sirius Red (SR) and birch Digital slide scanner (NanoZoomer S210, Hamamatsu Photonics, Shizuoka, Japan) The images were taken at ) . In addition, in Example B5, the images were taken at 2 weeks after cell administration (14 days) at the end of the experiment. Later, animals were sacrificed and their lungs were collected. The collected lungs were solidified with 4% paraformaldehyde. After determination, paraffin embedding was performed. The paraffin sections were Masson's Trichromium. After staining with e, imaging was performed using a BZ-X710 (Keyence, Osaka, Japan). The captured images... The fibrotic region was quantified using ImageJ software.

[0114] Primary culture of rat lung fibroblasts and fibroblasts with high ADM expression and enhanced VCAM-1 expression. Making > WKY / NCrlCrlj purchased from Charles River Japan (Kanagawa, Japan) Lung tissue was collected from a female rat (6 weeks old), and primary culture was performed to expand rat lung fibroblasts (rPF). Large-scale culture was performed. Specifically, rat lung tissue recovered by primary was cut with a scalpel to a size of 1 mm. Mince in the corner and add 0.14 Wunsch units / mL of Liberase™ Add Sigma Aldrich, St. Louis, MO) and incubate at 37°C. The cells were treated with enzymes in a tank and then cultured in DMEM (Low Glucose). ADM-highly expressing and VCAM-1-enhanced rPF (rVPF) is a type of mVPF described above. It was prepared using the same method as the manufacturing process. Specifically, rat recombinant TNF-α and IL were added to rPF. -4 (final concentration: 50ng / mL TNF-α, 2ng / mL IL-4), 7 The cells were prepared by culturing them for 2 hours. The VCAM-1 positive cell rate of rVPF was measured using MACSQu. ant Analyzer(Miltenyi Biotec,Bergisch Gl The results were evaluated by flow cytometry (adbach, Germany).

[0115] <Creation of a rat model of interstitial pneumonia and pulmonary fibrosis> The creation of disease model rats was commissioned to Charles River Japan (Kanagawa, Japan). N / CrlCrlj rats (male, 6 weeks old) were given bleomycin solution (5 mg / kg physiological diet). A single intratracheal spray of saline solution is administered using a liquid microsprayer, and interstitial pneumonia is treated. We created a pulmonary fibrosis model rat. The rat strain was selected for the following reasons. (1) BN rats and WKY rats are immunologically divergent in inbred strains, The selection of both lineages is widely used in efficacy testing of allogeneic cell therapies; (2) In this lineage There are reports of the creation of interstitial pneumonia and pulmonary fibrosis models (Non-patent document 17).

[0116] <Evaluation of the therapeutic effect of cell administration and 2 weeks after administration in rat models of interstitial pneumonia and pulmonary fibrosis> In interstitial pneumonia / pulmonary fibrosis model rats administered bleomycin solution 5 days later, the following species were used: Cells of the same type: 5.0 × 10 5 Administer intravenously as cells / 500 μL physiological saline / body. Each rat was given rPF, rVPF, or physiological saline (Ct) as a control. rl) was administered. The therapeutic effect of each cell was determined by survival analysis using the Kaplan-Meier method and pathological evaluation. The experiment was carried out. At the end of the experiment, two weeks (14 days) after cell administration, the animals were sacrificially killed and their lungs were collected. The collected lung tissue was fixed with 4% paraformaldehyde and then embedded in paraffin. The fin sections were stained with Masson's Trichrome and then BZ-X710(Ke The images were taken in Yence (Osaka, Japan). The captured images were processed using ImageJ software. The fibrotic region was quantified using method e.

[0117] <Culture of human lung fibroblasts and generation of VCAM-1-enhanced fibroblasts> Human adult lung fibroblasts are PromoCell (Heidelberg, Germany) Purchased from ) and added 5% (v / v) NBCS (Newborn Calf Serum) Fibroblast culture medium (HFDM-1(+)medium (Cell Science Institute Co., Ltd.) Large-scale culture was performed in Miyagi, Japan. To create fibroblasts with enhanced VCAM-1 expression, human lung fibroblasts are cultured in a cell culture dish. The cells were cultured in large quantities, and the group cultured without treatment (hPF) and the group cultured with TNF-α (50 ng / mL) (P eprotech, Cranbury, NJ) and IL-4 (2ng / mL) (Fujifilm) A group was established in which patients were treated with a two-drug mixture (hPF+CKs) from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All groups used HFDM-1(+) supplemented with 5% NBCS as the base medium for 3 days. (72 hours) The cells were cultured. hVPF was converted to hPF+CKs by anti-CD106(VCAM-1)-Bi otin, human antibody (Miltenyi Biotec, Bergisch Gl adbach, Germany) and Anti-Biotin microBeads (M Immunostaining was performed using iltenyi Biotec, and autoMACS pro Separ The cells were recovered by cell sorting using an ator (Miltenyi Biotec).

[0118] <Statistical analysis> Experimental results are expressed as mean ± standard deviation (SD). Significance testing was performed using the Kaplan-Meiei method. Survival analysis using the r method involves log-rank (Mantel-Cox) tests and log-rank trends. The results were calculated using statistical tests, and for other experiments, Student's t-test was used. P<0.05 is considered statistically significant. It was considered a difference. Furthermore, statistical analysis of fibrotic regions and cell characterization by flow cytometry were performed using Grap. hPad Prism 9 for Windows 64-bit,Version This was done using 9.2.0(332). In syngeneic transplantation experiments into mice, ordinary A one-way ANOVA was performed, and multiple cos were calculated based on Ctrl. Dunnett's multiple comparisons of mparisons This was performed in a test. In the allograft experiment in rats, ordinary one - A two-way ANOVA was performed, and multiple comparisons of each group were performed. The test was conducted using Ukey's multiple comparisons test. In the cellular characterization of lung fibroblasts, a 2-way ANOVA was performed, and mu for each group was determined. ltiple comparisons to Tukey's multiple comp The Arisons test was performed. In all trials, P<0.05 was considered statistically significant. That was my assessment.

[0119] <Example B1: Lymphangiogenic ability of fibroblasts with high ADM expression and enhanced VCAM-1 expression> Human adult-derived lung fibroblasts were cultured in cell culture dishes using adherent culture. Bright-field observation was performed. This indicates that cultured human adult-derived lung fibroblasts have a spindle-shaped fibroblast-like morphology. (Figure 9A) Cultured human adult-derived lung fibroblasts were treated with TNF-α (50 ng / mL) and IL-4 (2 ng / mL) was added as a two-component mixture (Factor-X) and incubated for 24 hours. Figure 9B) Flow cytometry (FCM) shows fibroblast markers CD90 and VCA The percentage of M-1 (CD106)-positive cells was evaluated (Figures 9C and 9D). Human adult-derived lung fibers. Almost all of the blast cells were CD90 positive, and untreated human adult-derived lung fibroblasts (Co The CD90-positive cell rate for ntrol was 96.00±4.33%, compared to TNF CD90 of human adult-derived lung fibroblasts (+Factor-X) supplemented with -α and IL-4 The positive cell rate was 95.56 ± 4.51%. Additionally, the CD106-positive cells in the control group... While the cell count was 6.03 ± 1.91%, the +Factor-X CD106-positive cells... The cytosis rate was 31.96 ± 5.78%. Both CD90 and CD106 positive (Double The +)) cell rate was 5.86±1.94% in the Control group, compared to +Fact or-X was 31.25±6.27%. From these results, TNF-α and IL-4 It was found that this significantly improved the percentage of CD106-positive cells in human lung fibroblasts. Next, human adult-derived lung fibers with improved CD106-positive cell rate due to Factor-X To evaluate the lymphangiogenic capacity of blast cells, VEGF-, a representative lymphangiogenic factor, is used. The gene expression levels of C and ADM were evaluated by RT-qPCR (Figure 9E). As a result, VE The gene expression levels of GF-C and ADM were increased to 2.99 each by the addition of Factor-X. Expression was ±0.39 times and 2.46±0.69 times higher, and a significant difference was observed between the two groups. Based on these results, we found that TNF-α and IL-4 improved VCAM-1 expression levels. Human lung fibroblasts highly express lymphangiogenesis genes and are rich in the ability to generate lymphangiogenesis in the lungs. It was suggested that it was a cyst.

[0120] <Example B2: Lymphangiogenesis effect of fibroblasts with high ADM expression and enhanced VCAM-1 expression> > Human-derived adult lung fibroblasts (VPF) with high ADM expression and enhanced VCAM-1 expression. To evaluate the lymphangiogenic effect, lymphatic endothelial cells recovered from human lung-derived cells were used. Human pulmonary microvascular endothelial cells were cultured on a large scale. Bright-field observation revealed that the cultured cells were laid on a bed. The cells exhibit a stone-like morphology (Figure 10A), and FCM analysis revealed that these cells are VE, an endothelial cell marker. - 99.71 ± 0.09% were positive for cadherin (N=3), and lymphatic endothelial cells were positive. It was revealed that 96.77 ± 0.37% were positive for the PDPN test (N=3 (Figure 10B). Endothelial cell marker VE-Cadherin and lymphatic endothelial cell marker The test was performed using lymphatic endothelial cells that were bipositive for PDPN, the marker for the test. Lymphatic endothelial cells recovered from human lung cells and various adult human lung fibroblasts are co-cultured. When the cells were cultured and the lymphatic vessels were observed by immunofluorescence staining, untreated adult-derived lung fibroblasts were found to be different from those of untreated adult-derived lung fibroblasts. While lymphatic vessels are fragmented and discontinuous vascular formation is observed, TNF-α (50 ng / mL) Adult-derived lung fibroblasts (+F) were cultured for 24 hours after adding IL-4 (2 ng / mL) and IL-4. It has been revealed that actor-X forms continuous blood vessels that are not interrupted. (Figure 10C). From these results, lung fibroblasts treated with TNF-α and IL-4 are linked to lymphatic vessels. It was revealed that these cells have a high regeneration potential.

[0121] Example B3: Interstitial pneumonia caused by fibroblasts with high ADM expression and enhanced VCAM-1 expression. Treatment effectiveness for pulmonary fibrosis > stroma of adult-derived lung fibroblasts treated with TNF-α and IL-4, which have a high lymphangiogenic effect. To evaluate the therapeutic effect of C57BL on pulmonary fibrosis, a syngeneic transplantation experiment was designed. / 6 Lung-derived fibroblasts were collected from adult mice and cultured in large quantities. Bright-field observation was performed. The cultured primary adult-derived lung fibroblasts exhibited a spindle-shaped fibroblast-like morphology. This is shown (Figure 11A). Cultured adult mouse lung fibroblasts were treated with TNF-α (50 ng / m³). L) and IL-4 (2 ng / mL) were added and cultured for 3 days, and adult mouse lungs were cultured. The percentage of CD106-positive fibroblasts was evaluated by FCM (Figure 11B). As a result, C Ontrol was 84.89±11.42% (N=3), while Factor- 96.52±3.32% of fibroblasts treated with X (N=3). From these results, Compared to human lung fibroblasts, Uss lung fibroblasts have a higher CD106-positive cell rate even under normal conditions. Although the levels are relatively high, TNF-α and IL-4 can improve the CD106-positive cell rate across species. That's what I found out. Mouse-derived adult lung fibroblasts (VPF) with high ADM expression and enhanced VCAM-1 expression. To evaluate the lymphangiogenic capacity of each adult-derived lung fibroblast, VEGF-C When the gene expression levels of ADM were evaluated by RT-qPCR, human adult-derived lung fibroblasts Similarly, the gene expression levels of VEGF-C and ADM were compared with TNF-α (50 ng / mL) and I The addition of L-4 (2 ng / mL) (Factor-X) resulted in values ​​of 4.11 ± 0.5, respectively. Expression was 9 times higher (N=3) and 1.70±0.27 times higher (N=3), with no significant difference between the two groups. This was observed (Figure 11C). From these results, mouse lung fibroblasts are similar to human lung fibroblasts. It has been revealed that TNF-α and IL-4 significantly increase the expression of lymphangiogenesis genes. This revealed that mouse-derived VPF cells are also cells rich in lymphangiogenesis ability.

[0122] Example B4: Interstitial pneumonia caused by fibroblasts with high ADM expression and enhanced VCAM-1 expression. Treatment effectiveness for pulmonary fibrosis > Adult-derived lung cancer with high ADM expression and enhanced VCAM-1 expression for interstitial pneumonia and pulmonary fibrosis To evaluate the therapeutic effect of fibroblasts (VPF), a mouse interstitial pneumonia / pulmonary fibrosis model was used (see figure). Cells were administered according to the experimental schedule described in 12A. The following three groups of model animals were established: Group administered adult Uss lung fibroblasts (+PFs, N=8), group administered mouse VPF (+VPF s (N=10), physiological saline administration group (Control, N=10). The effect on the prognosis of each group was evaluated using survival analysis with the Kaplan-Meier method. However, the survival rates on Day 28 were 40.00% (Control) and 37.50%, respectively. (+PFs), 80.00% (+VPFs) (Figure 12B). Also, median survival time These figures represent 14.5 days (Control), 14 days (+PFs), and 80% individual survival, respectively. Undefinable (+VPFs), and improvement in life expectancy was observed with VPF administration. Four weeks after cell administration, lung tissue was collected, and the lung tissue from each group was stained with Sirius Red for pathological observation. When the alveolar structure and the degree of fibrosis progression were evaluated, the Control group and the +PFs group showed Although no clear compartments of alveolar structure were observed, and fibrotic areas were confirmed mainly around the bronchi, Furthermore, the +VPFs group showed clear alveolar compartments, and no fibrotic areas were observed (Figure 12). C).

[0123] <Example B5: Administration of mVPF to a mouse interstitial pneumonia / pulmonary fibrosis model and the results after 2 weeks of administration> Evaluation of treatment effectiveness > Furthermore, in mouse interstitial pneumonia and pulmonary fibrosis model mice, mPF, mVPF, or When physiological saline (Ctrl) was administered and the survival rate two weeks after administration was compared, Ctr In group I, two deaths were confirmed on Day 10 and two on Day 14, resulting in a survival rate of 63.64%. (7 / 11 cases) (Figure 13A). On the other hand, in the mPF administration group, there was one case each on Day 7 and Day 9. One death was confirmed, and the survival rate was 71.43% (5 / 7 cases). Furthermore, mVPF administration In the treatment group, no deaths were confirmed, and the survival rate was 100% (5 / 5 cases). From these results... The study suggested that mVPF has a significant effect in improving survival rates for interstitial pneumonia and pulmonary fibrosis. Next, Masson's Trichrome staining of lung tissue collected 14 days after cell administration. Upon color analysis and pathological observation, the mVPF-treated group showed the least amount of fibrotic area. It was revealed that the alveolar structure was normal (Figure 13B). Furthermore, the fibrotic areas in each group were determined. In a dose-based evaluation, the mVPF administration group had the fewest fibrotic areas and, compared to the Ctrl group, It became clear that the region was surprisingly small (Figure 13C). Also, individuals on day 0 after cell administration and In comparison, the Ctrl group showed a significant increase in the fibrotic area, while the other groups (mPF-treated group) showed a significant increase in fibrotic area. No significant difference was observed in the mVPF administration group. Furthermore, compared to the mPF administration group, mV It was also found that the fibrotic area was smaller in the PF-treated group. These results suggest that mVPF is metastatic. Infusion and administration enhance the survival rate of bleomycin-induced interstitial pneumonia and pulmonary fibrosis in mouse models. It was found to have a function that suppresses the progression of fibrosis.

[0124] <Example B6: Isolation of rat lung fibroblasts and preparation of VCAM-1 positive rat lung fibroblasts> Made > Lung fibroblasts (rPF) were isolated from WKY / NCrlCrlj rats and DMEM(L Expanded culture was performed in (low Glucose) medium (Figure 14A). Next, rat cells were cultured in rPF. Add modified TNF-α (final concentration: 50 ng / mL) and IL-4 (final concentration: 2 ng / mL). In addition, we attempted to fabricate rVPF (Figure 14B). FCM analysis revealed that rPF is VCAM-1( The CD106)-positive cell rate was 25.18%, while TNF-α and IL-4 were added. The rVPF prepared using this method showed a positive cell rate of 80.02% (Figure 14C). Therefore, rP F, like mPF, shows responsiveness to TNF-α and IL-4, and rVPF can be fabricated. I understood that.

[0125] <Example B7: Administration of rVPF to a rat interstitial pneumonia / pulmonary fibrosis model and the results 2 weeks after administration> Evaluation of treatment effectiveness > Rat interstitial pneumonia and pulmonary fibrosis model mice were given rPF, rVPF, or physiological saline solution, respectively. When (Ctrl) was administered and the survival rate two weeks after administration was compared and evaluated, the rat model showed that Unlike the Uss model, all groups showed death or mortality two weeks after administration of cells or physiological saline. Since no such difference was observed, no difference in survival rates was found between the groups. On the other hand, Masson's Trichrome was used to examine lung tissue collected 14 days after cell administration. Staining and pathological observation revealed significant differences between the groups. The extensive fibrotic features observed in the lung (BLM) 5 days after syn administration and before cell administration were due to physiological saline. The condition worsens further with fluid administration (Ctrl), showing fibrosis and abnormal structural formation throughout the lung. This was found (Figures 15A and 15B). On the other hand, in the rPF and rVPF administration groups, fibrosis and Abnormal structural formation in the lungs was observed only locally, and the fibrotic area was significantly smaller than in Ctrl. There were no such results. Among them, the rVPF administration group showed a significant reduction in fibrotic areas compared to before cell administration. It was found to have an effect on improving the fibrotic area (Figures 15A and 15B). The results are similar to those of syngeneic transplantation experiments in mice, and VPF can be used as an autologous cell therapy as well as an allogeneic cell therapy. As a cellular drug, it was suggested to have ameliorative and preventive effects on pulmonary fibrosis.

[0126] <Example B8: Cellular characterization of human lung fibroblasts with enhanced VCAM-1 expression> Human adult-derived lung fibroblasts (hPF) were cultured in cell culture dishes using adherent culture. The cultured hPF was mixed with two reagents: TNF-α (50 ng / mL) and IL-4 (2 ng / mL). (Factor-X) was added and incubated for 72 hours to produce hPF+CKs. CKs were sorted using VCAM-1 (CD106) as an indicator, and an hVPF was created. . Flow cytometry (FCM) shows fibroblast markers CD90 and VCAM-1. The percentage of CD106-positive cells was evaluated (Figure 16A). Human adult-derived lung fibroblasts were almost entirely positive. All cells were positive for CD90, and the CD90-positive cell rate of hPF was 99.68 ± 0.06%. While the N=5 group had a CD90-positive cell rate in hPF+CKs, the rate was 98.81±0.5. The percentage was 1% (N=5). Furthermore, the CD90-positive cell rate in hVPF was 99.08±0.41. The percentage was % (N=5), and no significant differences were observed between the groups (Figure 16B). On the other hand, the percentage of VCAM-1 (CD106) positive cells in hPF was 15.49 ± 2.52%. While the percentage of CD106-positive cells in hPF+CKs was 5 (N=5), the percentage of CD106-positive cells in hPF+CKs was 31.38±3. The percentage was 41% (N=5). Furthermore, the CD106-positive cell rate in hVPF was 98.11±1. 0.1% (N=5) showed that hPF+CKs had a significantly higher CD106-positive cell rate than hPF. Furthermore, it was found that hVPF had an even more significantly higher percentage of positive cells than hPF+CKs (Figure). 16B). The percentage of cells positive for both CD90 and CD106 (DP) was similar, with the DP for hPF being 15.38± While the DP for hPF+CKs was 2.59% (N=5), the DP for hPF+CKs was 31.21±3.30. The percentage was % (N=5). Also, the DP of hVPF was 97.47±1.18% (N=5). Furthermore, hPF+CKs had significantly higher DP than hPF, and hVPF had a higher DP than hPF+CKs. Furthermore, it was found that DP was significantly higher (Figure 16B).

[0127] Based on the above, fibroblasts treated with TNF-α and IL-4 are fibroblast ADM To improve the percentage of highly expressed and VCAM-1-positive cells, and also to improve the percentage of highly expressed ADM and VCAM-1 Fibroblasts with enhanced expression of [specific gene] have improved lymphangiogenesis ability, thus creating a continuous network of lymphatic vessels. It was revealed that it promotes tubule formation. And the fibroblasts in question, especially those derived from the lungs, Intravenous administration of blast cells from the same lineage significantly improved the prognosis of mice with interstitial pneumonia and pulmonary fibrosis. Furthermore, it was revealed that these cells are effective in improving fibrosis. Adult-derived fibroblasts with enhanced VCAM-1 expression, particularly VPF, are also used as autologous cell therapies. As a cell therapy, it has been suggested to have ameliorative and preventive effects on pulmonary fibrosis. Therefore, AD Adult-derived fibroblasts with high M expression and enhanced VCAM-1 expression, especially those administered intravenously with VPF, improve prognosis. It is expected to be a novel treatment for pulmonary fibrosis, including IPF, which is known to be a serious disease. Cut.

Claims

1. A pharmaceutical composition comprising fibroblasts with high expression of ADM and / or HHEX for improving a condition of reduced lymphangiogenic ability, wherein the gene expression level of ADM and / or HHEX in the cells is 1.40 times or more higher than that in control fibroblasts.

2. A pharmaceutical composition comprising fibroblasts with high expression of ADM and / or HHEX for improving disorders of tissue fluid homeostasis, wherein the gene expression level of ADM and / or HHEX in the cells is 1.40 times or more higher than that in control fibroblasts.

3. A pharmaceutical composition for reducing edema, comprising fibroblasts with high expression of ADM and / or HHEX, wherein the gene expression level of ADM and / or HHEX in the cells is 1.40 times or more higher than that in control fibroblasts.

4. A pharmaceutical composition comprising fibroblasts with high expression of ADM and / or HHEX for improving impaired lipid and / or vitamin transport ability, wherein the gene expression level of ADM and / or HHEX in the cells is 1.40 times or more higher than that in control fibroblasts.

5. A pharmaceutical composition comprising fibroblasts with high expression of ADM and / or HHEX for activating the immune surveillance mechanism, wherein the gene expression level of ADM and / or HHEX in the cells is 1.40 times or more higher than that of a control fibroblast.

6. The pharmaceutical composition described in any one of claims 1 to 5, wherein the fibroblasts are fibroblasts of adult origin.

7. A pharmaceutical composition according to any one of claims 1 to 6 for treating fibrosis.

8. The pharmaceutical composition according to any one of claims 1 to 7, which is an injectable composition.

9. The pharmaceutical composition according to any one of claims 1 to 7, wherein the cells are constructed as a planar or three-dimensional cell tissue.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the fibroblasts are cardiac-derived fibroblasts.

11. To treat lung diseases, fibroblasts with high ADM expression; A fibroblast population comprising the ADM-highly expressing fibroblasts; or A cell population comprising CD106-positive lung-derived fibroblasts, wherein the percentage (based on cell number) of the CD106-positive lung-derived fibroblasts is more than 18.01% of the total fibroblasts contained in the cell population. A pharmaceutical composition comprising: The ADM gene expression level of the ADM highly expressing fibroblasts is 1.40 times or more higher than that of a control fibroblast; The pharmaceutical composition, wherein the lung disease is pulmonary fibrosis or interstitial pneumonia.

12. To improve the condition of decreased lymphangiogenesis, fibroblasts with high ADM expression; A fibroblast population comprising the ADM-highly expressing fibroblasts; or A cell population comprising CD106-positive lung-derived fibroblasts, wherein the percentage (based on cell number) of the CD106-positive lung-derived fibroblasts is more than 18.01% of the total fibroblasts contained in the cell population. A pharmaceutical composition comprising: A pharmaceutical composition, wherein the ADM gene expression level in the ADM highly expressing fibroblasts is 1.40 times or more higher than that in a control fibroblast.

13. The pharmaceutical composition described in claim 11 or 12, wherein the fibroblasts with high ADM expression have high VEGF-C expression, and the gene expression level of VEGF-C in the fibroblasts with high ADM expression is 1.40 times or more higher than that in a control fibroblast.

14. The pharmaceutical composition described in any one of claims 11 to 13, wherein the fibroblasts highly expressing ADM are CD106 positive.

15. The pharmaceutical composition described in any one of claims 11 to 14, wherein the fibroblasts highly expressing ADM are lung-derived fibroblasts.

16. The pharmaceutical composition according to any one of claims 11 to 15, which is an injectable composition.

17. The pharmaceutical composition according to any one of claims 11 to 15, wherein the cell population is constructed as a cell tissue having a planar or three-dimensional shape.

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