Application of matrix-enabled mesenchymal stem cells

Abstract

The invention relates to the technical field of biology, in particular to application of matrix-enabled mesenchymal stem cells, the matrix-enabled mesenchymal stem cells are obtained by culturing and enabling mesenchymal stem cells on a cell culture medium hENFc, and the cell culture medium hENFc is prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc; matrix-enabled mesenchymal stem cells can promote cell nucleus transfer of beta-catenin, enhance multiplication capacity and stemness maintenance capacity of in-vitro amplification and delay cell senescence and apoptosis, and hMSCs subjected to long-term enabled culture (fifteenth generation and above) of hENFc do not have tumorigenicity, do not have any adverse effect on animals and meet the clinical application standard of hMSCs; the traditional Chinese medicine composition can be used for treating immune disorder diseases, such as systemic lupus erythematosus, diabetic nephropathy, systemic sclerosis and xerosis, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to the application of matrix-enabled mesenchymal stem cells. Background Art

[0002] Immune diseases refer to clinical symptoms caused by the body's self-immune tolerance being broken or autoimmune cell regulation being abnormal under the induction of certain internal and external factors such as genetic factors and environmental factors, resulting in a persistent and prolonged immune response to self-antigens, causing damage to self-tissue cells or dysfunction. Common immune diseases include systemic lupus erythematosus, systemic sclerosis, Sjögren's disease, hyperthyroidism, juvenile diabetes, diabetic nephropathy, idiopathic thrombocytic purpura, autoimmune hemolytic anemia, lupus nephritis, rheumatoid arthritis, systemic vasculitis, ulcerative colitis, chronic hepatitis, polyneuritis, etc. Immune diseases are currently mainly treated with drugs. Commonly used drugs include immunosuppressants, the use of antibodies or blockers against cytokines and their receptors, the use of antibodies against immune cell surface molecules, and the induction of immune tolerance through oral administration of self-antigens.

[0003] Stem cells have properties such as self-renewal and the ability to generate cells of different lineages, which have led to their extensive exploration in cell therapies for the treatment of various human diseases. Due to their regenerative properties, ability to secrete trophic factors and multi-lineage differentiation, and lack of ethical restrictions, stem cells have become an important source of stem cells in regenerative medicine. In addition, stem cells also exhibit low immunogenicity, homing effects, and immunomodulatory functions, and have become the most common cell source in preclinical and clinical studies by affecting the microenvironment of damaged tissues and enhancing tissue repair. Currently, stem cells can treat a range of diseases by restoring organ homeostasis in inflamed, injured, or diseased tissues.

[0004] Stem cell therapy for immune diseases has been used clinically. For example, Li Yuehua et al. found that adipose-derived mesenchymal stem cells can be used clinically to treat autoimmune diseases. [1] Shu Zhaoping et al. found that mesenchymal stem cells can be used to treat systemic lupus erythematosus [2] However, due to the characteristics of mesenchymal stem cells, the quality of MSCs will decrease with the increase of in vitro culture time, because continuous subculture will lead to changes in cell morphology, cell enlargement and eventual aging; the homing effect of MSCs after in vitro expansion is significantly reduced, and the expression pattern changes significantly; traditional culture expansion will gradually reduce the therapeutic efficacy of MSCs, etc., which seriously affects the clinical application of MSCs. In addition, it is difficult to predict the immunomodulatory and regenerative effects of MSCs in human trials.

[0005] Therefore, providing a mesenchymal stem cell that can promote the nuclear transfer of β-catenin, enhance the proliferation ability of in vitro expansion, maintain stemness, delay cell aging and apoptosis, and has no tumorigenicity after long-term empowerment culture of hENFc (eleventh generation and above), and has no adverse effects on animals, and meets the clinical application standards of hMSCs has become a technical problem that needs to be solved by those skilled in the art. .

[0006] In view of the above technical problems, the present invention provides a new type of mesenchymal stem cells, wherein the matrix-enabled mesenchymal stem cells are obtained by culturing and enabling mesenchymal stem cells in a cell culture matrix hENFc, wherein the cell culture matrix hENFc is prepared from 1-3 parts of hN-cad-Fc and 1-3 parts of hN-cad-Fc. The matrix-enabled mesenchymal stem cells can promote the nuclear transfer of β-catenin, enhance the proliferation ability of in vitro expansion, the ability to maintain stemness, and delay cell aging and apoptosis. The hMSCs cultured to the fifteenth generation through hENFc empowerment are not tumorigenic and have no adverse effects on animals, which meets the clinical application standards of hMSCs. The matrix-enabled mesenchymal stem cells are used to treat immune diseases and obtain good therapeutic effects.

[0007] References:

[0008] [1] Li Yuehua, Li Qingfeng, Xie Yun. Application progress of adipose-derived mesenchymal stem cells in autoimmune diseases[J]. Journal of Shanghai Jiaotong University (Medical Edition), 2022, 42(08): 1131-1138.

[0009] [2] Shu Zhaoping, Yuan Yuan, Fu Qiang. Mechanism and research progress of mesenchymal stem cells in the treatment of systemic lupus erythematosus[J]. Journal of Guangzhou Medical University, 2024, 52(02): 75-80. Summary of the invention

[0010] The primary purpose of the present invention is to provide an application of matrix-enabled mesenchymal stem cells in the preparation of drugs for preventing and treating immune diseases. The matrix-enabled mesenchymal stem cells are obtained by culturing and enabling mesenchymal stem cells in a cell culture matrix hENFc, and the cell culture matrix hENFc is prepared from 1-3 parts of hN-cad-Fc and 1-3 parts of hE-cad-Fc.

[0011] Preferably, the cell culture matrix hENFc is diluted with hE-cad-Fc and hN-cad-Fc to a total protein concentration of 1-30 μg / mL, added to a tissue culture treated plate, incubated for 0.5-24 hrs, and washed.

[0012] Preferably, the immune diseases include autoimmune diseases and inflammatory immune diseases.

[0013] Preferably, the autoimmune diseases include one or more of systemic lupus erythematosus, systemic sclerosis, Sjögren's disease, hyperthyroidism, juvenile diabetes, diabetic nephropathy, idiopathic thrombocytic purpura, and autoimmune hemolytic anemia; and the inflammatory immune diseases include one or more of lupus nephritis, rheumatoid arthritis, systemic vasculitis, ulcerative colitis, chronic hepatitis, and polyneuritis.

[0014] Preferably, the mesenchymal stem cells are derived from mammals.

[0015] Preferably, the mesenchymal stem cells are derived from humans, pigs or mice.

[0016] Preferably, the mesenchymal stem cells are derived from bone marrow, adipose tissue, dental pulp, umbilical cord, umbilical cord blood, amniotic fluid or placenta.

[0017] Preferably, the mesenchymal stem cells are derived from umbilical cord or umbilical cord blood.

[0018] The beneficial effects of the present invention are as follows: (1) The present invention provides an application of matrix-enabled mesenchymal stem cells in the preparation of drugs for preventing and treating immune diseases, wherein the mesenchymal stem cells are obtained by culturing and enabling a cell culture matrix hENFc, wherein the cell culture matrix hENFc is prepared from 1-3 parts of hN-cad-Fc and 1-3 parts of hE-cad-Fc, and the matrix-enabled mesenchymal stem cells can promote the nuclear transfer of β-catenin, enhance the proliferation ability of in vitro expansion, the ability to maintain stemness, and delay cell aging and apoptosis, and hMSCs cultured to the eleventh generation through hENFc empowerment are not tumorigenic and have no adverse effects on animals, thus meeting the clinical application standards of hMSCs.

[0019] (2) The matrix-empowered mesenchymal stem cells not only have better survival ability in the lesion microenvironment, but also have stronger environmental perception and anti-inflammatory ability, and significantly inhibit PBMC proliferation ( Figure 5 A) and promote cell apoptosis ( Figure 5 B), MSC EN It also suppressed the proportion of CD4+ and CD8+ T cell subsets, improving the efficacy of hMSCs in inflammatory and autoimmune diseases.

[0020] (3) The matrix-empowered mesenchymal stem cells described above can treat systemic lupus erythematosus. The biochemical indexes and immune organ pathological indexes of the treated mice were significantly improved, and the thickening of the glomerular mesangium and basement membrane, inflammatory cell infiltration, interstitial fibrosis and occasional crescent formation were significantly reduced;

[0021] (4) The matrix-empowered mesenchymal stem cells can treat diabetic nephropathy. The pathological symptoms of the renal tissue of the treated mice were alleviated, and the progression of diabetic nephropathy was significantly delayed. The increase of blood glucose, 24-hour urine protein, BUN and SCr in rats was significantly inhibited. The potential of hMSCs to slow down kidney damage in diabetic rats was effectively improved, the renal function of DKD rats was improved, the cell damage caused by oxidative stress was effectively reduced, the development of diabetic nephropathy was alleviated, and it had a stronger anti-apoptosis ability.

[0022] (5) The matrix-empowered mesenchymal stem cells can inhibit the proliferation of activated T cells. EN Compared with the MSC group cultured in TCPS, the cells had an inhibitory efficiency of about 30%, effectively reversed the fibrotic phenotype of skin fibroblasts, inhibited the expression of fibrosis-related genes, downregulated Col1A1 by about 25% and α-SMA by about 17%; slowed down the immune infiltration of systemic sclerosis lesions (pro-inflammatory CD4+ lymphocytes decreased by 6.5%, Th1 lymphocytes were inhibited by 0.17%, and anti-inflammatory regulatory T lymphocytes Treg cells were enhanced by 0.2%); reversed the degree of fibrosis of skin fibroblasts (the thickness of the dermis decreased by about 20%, and the thickness of the subcutaneous fat layer increased by about 1.5 times); effectively reduced collagen deposition in skin and lung tissues, and reversed the degree of tissue fibrosis (downregulated COL1A1 and COL1A2 by about 30%, downregulated COL3A1 by about 25%, and α-SMA by about 20%); and significantly improved the vascular integrity and permeability of systemic sclerosis.

[0023] (6) The matrix-empowered mesenchymal stem cells can improve the salivary gland function of Sjögren's disease, upregulate salivary gland flow rate, reduce inflammatory cell infiltration, reduce atrophy of alveoli and glandular ducts, reduce the degree of salivary gland fibrosis, increase the expression of aquaporin, and improve the salivary gland function of Sjögren's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings illustrate the disclosed embodiments and serve to explain the principles of the disclosed embodiments. However, it is to be understood that the accompanying drawings are designed only for illustration purposes and not as a definition of the limits of the present invention.

[0025] Figure 1 Preparation of hENFc

[0026] Note: (A) The fixed amount-protein concentration curve of hE-cad-Fc and hN-cad-Fc; (B) The fixed amount-time curve of hE-cad-Fc and hN-cad-Fc; (C) The interaction curve between hE-cad-Fc and hN-cad-Fc; (D) The fixed amount detection of hE-cad-Fc and hN-cad-Fc on different matrix surfaces

[0027] Figure 2 Characterization of hENFc

[0028] Note: (A) Element analysis on the surface of different matrices; (B) Stability detection of different matrices; (C) Distribution and structure detection of hE-cad-Fc and hN-cad-Fc on the surface of different matrices; (D) Hydrophilicity detection of different matrices

[0029] Figure 3 Detection of the proliferation ability of hMSCs at different passages on different matrix surfaces

[0030] Note: (A) cell doubling time; (B) cell number

[0031] Figure 4 Statistics of hMSCs volume at different generations on different matrix surfaces

[0032] Figure 5 Detection of endogenous cadherin expression in hMSCs of different passages on different matrix surfaces

[0033] Note: (A) Fluorescence quantitative PCR detection; (B) Western Blotting detection

[0034] Figure 6 Nuclear and cytoplasmic distribution of β-catenin protein in hMSCs on different matrix surfaces

[0035] Figure 7 Expression of OCT4 gene and protein in hMSCs of different passages on different matrices

[0036] Figure 8 Senescence detection of hMSCs at different passages on different matrix surfaces

[0037] Note: (A) Cell cycle detection; (B) β-galactosidase staining

[0038] Fig. 9 Apoptosis detection of hMSCs at different passages on different matrices

[0039] Fig.10 Multiple differentiation ability tests of hMSCs at different passages on different matrix surfaces

[0040] Note: (A) Oil red O staining and adipogenic gene expression detection; (B) Alizarin red staining and osteogenic gene expression detection; (C) Alcian blue staining and chondrogenic gene expression detection

[0041] Fig.11 MSC EN Oncogene and tumor suppressor gene expression dot plots

[0042] Fig.12 MSC EN Karyotype analysis

[0043] Fig.13 MSC EN Tumorigenicity assay

[0044] Note: (A) Light microscopic image of mouse tumor size; (B) Mouse weight; (C) Tumor volume

[0045] Fig.14 Effect of IFN-γ stimulation on MSCs EN The impact of vitality

[0046] Note: (A) Cell viability detection; (B) Western Blotting detection and quantitative statistics of apoptosis-related proteins

[0047] Fig.15 Effect of IFNγ stimulation on MSCs EN Impact of anti-inflammatory ability

[0048] Note: (A) Fluorescence quantitative PCR and Western Blotting detection and quantitative statistics of IDO expression; (B) Elisa detection of sTNFR1 release

[0049] Fig.16 MSC EN Effects of co-culture on proliferation and differentiation of PBMC

[0050] Note: (A) PBMC proliferation; (B) PBMC apoptosis; (C) CD4+T cell ratio; (D) CD8+T cell ratio

[0051] Fig.17 MSC EN Effects of co-culture on CD4+T cell subtypes in PBMC cells

[0052] Note: (A) The proportion of Th1 cells (CD4+IFN-γ+) in CD4+PBMC; (B) The proportion of Th2 cells (CD4+IL4+) in CD4+PBMC; (C) The proportion of Th17 cells (CD4+IL17A+) in CD4+PBMC; (D) The proportion of Treg cells (CD4+CD25+FoxP3+) in CD4+PBMC

[0053] Fig.18 MSC EN Elisa detection of inflammatory factors in the co-culture system with PBMC

[0054] Fig.19 MSC EN Research protocol for treating lupus nephritis in mice

[0055] Fig. 20 MSC ENEffects of transplantation on physiological and biochemical parameters of mice

[0056] Note: (A) Mouse weight; (B) Urine protein content; (C) ds-DNA content in blood; (D) TNF-α content in blood

[0057] Fig.21 Pathological staining of mouse kidneys 2 weeks after transplantation

[0058] Note: (A) H&E staining; (B) PAS staining; (C) Masson staining

[0059] Fig. 22 MSC EN Renal pathology scores of mice 2 weeks after transplantation

[0060] Fig.23 Immunofluorescence staining of IgG and C3 in mouse kidneys 2 weeks after hMSCs transplantation

[0061] Fig.24 MSC EN Analysis of immune cell status in perinephric lymph nodes of mice 2 weeks after transplantation

[0062] Note: (A) CD4+T cell analysis; (B) CD8+T cell analysis

[0063] Fig.25 MSC EN Analysis of CD4+T cell subtypes in perinephric lymph nodes of mice 2 weeks after transplantation

[0064] Fig.26 MSC EN Analysis of immune cell status in mouse spleen 2 weeks after transplantation

[0065] Note: CD4+T cell analysis; (B) CD8+T cell analysis;

[0066] Fig. 27 MSC EN Analysis of CD4+T cell subtypes in mouse spleen 2 weeks after transplantation

[0067] Fig.28 Construction of diabetic nephropathy model in rats and MSC EN Treatment options

[0068] Fig.29 MSC EN Effects of transplantation on physiological and biochemical parameters in rats

[0069] Note: (A) 24h water intake; (B) 24h food intake; (C) rat body weight; (D) blood sugar; (E) 24h urine protein; (F) BUN content; (G) serum creatinine SCr content

[0070] Fig.30 MSC EN Effects of transplantation on rat kidneys 2 weeks after transplantation

[0071] Note: (A) Kidney weight; (B) Light microscopic image of kidney; (C) Kidney index

[0072] Fig.31 MSC EN Pathological staining of rat kidney 2 weeks after transplantation

[0073] Fig.32 MSC EN Detection of oxidative stress level in rat kidneys 2 weeks after transplantation

[0074] Fig.33 MSC EN Effects of 2-week transplantation on renal apoptosis in rats

[0075] Note: (A) Western Blotting detection and quantitative statistics of NRF2 and HO-1 expression; (B) Immunohistochemical staining of NRF2

[0076] Fig.34 MSC EN Western Blotting Detection and Quantitative Statistics of Apoptosis-related Protein Expression in Rat Kidneys 2 Weeks After Transplantation

[0077] Fig.35 MSC EN Effects of transplantation for 2 weeks on systemic inflammation in rats

[0078] Note: (A) Spleen weight; (B) Spleen observation light microscopy; (C) Spleen index; (C) Fluorescence quantitative PCR detection of pro-inflammatory cytokines in kidney; (C) Elisa detection of pro-inflammatory cytokines in serum

[0079] Fig.36 MSC EN Effects of transplantation on renal function in rats after 2 weeks Note: (A) Immunofluorescence staining of Synaptopodin protein; (B) Western Blotting detection and quantitative statistics of Nephrin and WT1 expression

[0080] Fig.37 MSC EN Cell inhibition of PBMC proliferation

[0081] Fig.38 MSC EN Cells inhibit fibrosis

[0082] Fig.39 MSC EN Inhibits inflammatory infiltration

[0083] Fig.40 MSC EN Cells inhibit skin fibrosis

[0084] Fig.41 MSC EN Cells inhibit skin fibrosis

[0085] Fig.42 MSC EN Cells improve vascular permeability

[0086] Fig.43 MSC EN Treatment of Sjogren's disease

[0087] (A) Salivary gland flow rate; (B) Salivary gland morphology; (C) Salivary gland / body weight ratio; (D) Salivary gland pathological evaluation (H&E staining, glycogen staining and Masson staining); (E) Salivary gland fluorescence staining (aquaporin AQP5 and ion transporter NKCC1) DETAILED DESCRIPTION

[0088] Non-limiting embodiments of the present invention will be further described in greater detail by reference to specific examples, which should not be construed as limiting the scope of the present invention in any way.

[0089] In the following examples, unless otherwise specified, all reagents used can be purchased from the market.

[0090] In the following examples, unless otherwise specified, the methods used are all conventional methods.

[0091] In the following embodiments, the hE-cad-Fc modified matrix is ​​referred to as hEFc, and the hN-cad-Fc modified matrix is ​​referred to as hNFc, wherein hE-cad-Fc and hN-cad-Fc have been disclosed in the invention patents CN109337857B "Use of fusion proteins E-cadherin-Fc, VE-cadherin-Fc and VEGF-Fc" and CN115040695B "Application of a fusion protein active interface based on VE-cad-Fc / N-cad-Fc" previously applied by the inventor, respectively, and the corresponding sequences are disclosed in detail, which will not be listed in this patent.

[0092] The matrix jointly modified by hE-cad-Fc and hN-cad-Fc is referred to as hENFc.

[0093] All data are expressed as mean ± standard deviation; statistical differences among multiple groups were evaluated by one-way analysis of variance (ANOVA). p<0.05 was considered statistically significant, *p<0.05, **p<0.01, ***p<0.001, ns indicated no significant difference. Unless otherwise specified, all in vitro experimental results were from 3 independent parallel experiments, and all animal experimental results were from 5 independent parallel experiments. All results were statistically analyzed using GraphPad Prism version 8.0 software.

[0094] Example 1. Preparation of hENFc matrix and matrix-empowered mesenchymal stem cells

[0095] 1. Preparation of hENFc Matrix

[0096] Purified hE-cad-Fc and / or hN-cad-Fc were diluted in PBS to a total protein concentration range of 0-30 μg / mL, and 150 μL of protein solution per square centimeter was added to a tissue culture treated plate (TCPS); after incubation at 37°C for 30-150 min, the matrix was further washed 5 times with PBS.

[0097] 2. Detection of adsorption amount-concentration and adsorption amount-time of hE-cad-Fc and hN-cad-Fc

[0098] To evaluate the amount-concentration relationship of hE / N-cad-Fc immobilized on TCPS, purified hE-cad-Fc or hN-cad-Fc was first diluted in PBS to reach a concentration range of 0, 1, 3, 5, 10, 20, and 30 μg / mL, and 100 μL was added to each well of a 96-well plate. After incubation at 37°C for 2 hrs, the plate was further washed 3 times with PBS. To evaluate the amount-time relationship of hE / N-cad-Fc immobilized on TCPS, purified hE-cad-Fc or hN-cad-Fc was first diluted in PBS to 5 μg / mL, and 100 μL was added to each well of a 96-well plate. After incubation at 37°C for 0, 30, 60, 90, 120, 150, and 180 min, the plate was further washed 3 times with PBS. Finally, the modified surface was determined by enzyme-linked immunosorbent assay (ELISA). Briefly, each substrate was incubated with 1.0% (w / v) bovine serum albumin (BSA) for 1 h to block nonspecific interactions, and then incubated with mouse anti-E-cadherin antibody (extracellular domain, 1:2000 dilution) or rabbit anti-N-cadherin antibody (extracellular domain, 1:2000 dilution) at 37°C for 2 hrs; after washing three times with PBS, the modified surface was incubated with HRP-labeled goat anti-rabbit / mouse IgG (H+L) antibody (1:5000 dilution) for 2 hrs, and then TMB solution was added and incubated at 37°C in the dark; after 15 min, stop buffer was added, and the absorbance was measured at 450 nm using a microplate reader.

[0099] 3. Hydrophilicity detection, elemental analysis and morphology detection of hENFc matrix

[0100] The prepared TCPS, hEFc, hNFc and hENFc substrate surfaces were washed with ultrapure water for 5 times and then dried. The surface hydrophilicity of different surfaces was evaluated using a water contact angle (WCA) meter. The elemental changes before and after substrate surface modification were evaluated using an X-ray photoelectron spectroscopy (XPS) device using a monochromatic MgKα radiation source (1253.6eV). C1s (binding energy 284.8eV) was used as a reference for charge correction, and the high-resolution spectra of C1s, N1s, O1s and S2p were analyzed using Avantage software. The morphology and roughness of the modified surface were characterized by atomic force microscopy (AFM).

[0101] 4. Stability test of hENFc matrix

[0102] Purified hE-cad-Fc and hN-cad-Fc were diluted in PBS to a total concentration of 5 μg / mL, and 100 μL per well was added to a 96-well plate. After incubation at 37°C for 2 hrs, the plate was further washed 3 times with PBS. Finally, the modified surface was determined by Elisa. Briefly, each matrix was incubated with 1.0% (w / v) BSA for 1 h to block nonspecific interactions, and then incubated with goat anti-human IgG (H+L) antibody (1:5000 dilution) for 2 hrs, and then TMB solution was added and incubated at 37°C in the dark. After 15 min, stop buffer was added and the absorbance was measured at 450 nm using an ELISA reader.

[0103] 6. Results

[0104] 6.1 Identification of hN-cad-Fc, hE-cad--Fc and preparation of hENFc

[0105] like Figure 1 As shown in A, the hE-cad-Fc and hN-cad-Fc immobilized on the TCPS surface are related to the original solution concentration. When the Cad-Fc concentration is lower than 5 μg / mL, the surface protein immobilization amount is proportional to the concentration and reaches saturation when the Cad-Fc concentration is 10 μg / mL. Figure 2 B proves that the amount of surface protein immobilization is also affected by the fixation time, and the maximum amount of protein immobilization is reached when incubated for 120 minutes. Figure 1 C shows that fusion protein matrices with different ratios of hE-cad-Fc and hN-cad-Fc can be prepared by controlling the Cad-Fc concentration and fixation time. In order to avoid the interaction between the two Cad-Fc in solution, I first observed through micro-thermophoresis experiments that the minimum concentration of the two Cad-Fc interactions was 400μg / mL, which was much greater than the saturated fixed amount of protein solution concentration (10μg / mL). In order to construct cadherin fusion protein matrices with different subtypes and ratios, we kept the total concentration of hE-cad-Fc and hN-cad-Fc constant at 5μg / mL, while changing their ratio (hE-cad-Fc: hN-cad-Fc = 2:1, 1:1 and 1:2) to prepare hEFc, hNFc and hENFc matrices with different Cad-Fc ratios ( Figure 1 D).

[0106] 6.2 Physicochemical Characterization of hENFc Matrix

[0107] XPS analysis showed that the N1s element content increased significantly after Cad-Fc surface modification, indicating that Cad-Fc was successfully immobilized on the TCPS surface ( Figure 2 A). Figure 2B shows that these Cad-Fc matrices can be stably maintained in the culture medium for 5 days. The morphological analysis of these matrices by AFM showed that nanorod-like structures of about 5-7 nm can be clearly observed on the surface of the TCPS plate ( Figure 2 C) Figure 2 As shown in Figure D, the hydrophilicity of these Cad-Fc matrices is significantly higher than that of TCPS. Cad-Fc was effectively immobilized on the TCPS plate and enhanced the hydrophilicity of the culture plate surface.

[0108] The inventors studied the effect of hENFc matrix on the adhesion and proliferation of hMSCs. As the proportion of hE-cad-Fc in the cadherin fusion protein matrix increased, the expression of OCT4 in hMSCs increased. Among them, when the proportion of hE-cad-Fc was greater than or equal to hN-cad-Fc, the level of OCT4 gene expression in hMSCs was significantly higher than that of hMSCs cultured on the TCPS surface, and its expression level no longer increased with the increase of the proportion of hE-cad-Fc. Therefore, the hENFc composite matrix (hE-cad-Fc: hN-cad-Fc = 1:1) was selected as the preferred matrix for subsequent research.

[0109] Example 2: Preparation and function of hENFc matrix-enabled hMSCs

[0110] 1. Preparation of hENFc matrix-enhanced hMSCs

[0111] First, hMSCs were cultured on TCPS to the second generation; then hMSCs were cultured at 1×10 6 The cells were seeded into T-75 cell culture flasks with hEFc, hNFc or hENFc matrix and cultured continuously to the 15th generation under conventional expansion conditions to obtain empowered hMSCs of different generations.

[0112] In the following experiments, hMSCs of each generation were prepared using unmodified TCPS matrix as a control. E hNFc-enhanced hMSCs are referred to as MSCs N hENFc matrix-enabled hMSCs are referred to as MSCs. EN hMSCs cultured normally on TCPS matrix are referred to as MSCs. T .

[0113] 2. Detection of the proliferation ability of hENFc matrix-enabled hMSCs

[0114] All cells collected on the surface of different matrices at the end of each passage were counted after excluding dead cells using trypan blue. Another way is to inoculate MSCs into TCPS, hEFc, hNFc or hENFc matrices and use CCK-8 to measure cell viability after incubation for 24 and 48 hours. In order to compare the cell proliferation rate at different passages, the doubling time (DT) and cell population doubling level (PDL) were calculated according to the following formula:

[0115]

[0116] Where N(t) is the number of cells at the time of subculture, N(to) is the number of cells seeded at the previous subculture, A(48) is the cell absorbance value at 48 h, and A(24) is the cell absorbance value at 24 h.

[0117] 3. Detection of nuclear and cytoplasmic distribution of β-catenin

[0118] hMSCs were seeded at a density of 1×104 cells per square centimeter on the surface of six-well TCPS, hEFc, hNFc or hENFc matrix and cultured for 2 hours, and then cell membrane proteins and nuclear proteins were extracted: cells were digested with EDTA, and the precipitate was collected by centrifugation at 500×g for 3 minutes. 200μL of plasma protein extraction reagent was added to each sample and vortexed at high speed for 15 seconds. After ice bathing for 10 minutes, centrifugation was carried out at 12000×g at 4℃ for 10 minutes. After the supernatant was removed, 100μL of nuclear protein extraction reagent was added, vortexed for 15 seconds, ice bathed for 10 minutes, and centrifuged at 16000×g at 4℃ for 10 minutes to obtain nuclear proteins; the expression and distribution of β-catenin protein were detected by Western Blotting.

[0119] 4. Detection of stemness of hMSCs empowered by hENFc matrix

[0120] MSCs were harvested at passages 6, 11, and 15. T and MSC EN Total RNA was extracted by the method of Example 2, and total protein was extracted by the method of Example 2; and the expression of E-cadherin, N-cadherin and OCT4 genes and proteins was detected by fluorescent quantitative PCR and Western Blotting.

[0121] The following primers were used for PCR detection of relevant gene expression:

[0122]

[0123] 5. Cycle detection of hENFc matrix-enhanced hMSCs

[0124] At the 6th, 11th and 15th passages, 1×10 6 MSC T and MSC EN The cells were fixed in 70% cold ethanol for 2 h to overnight; after washing with PBS three times, the cells were centrifuged at 1000 rpm for 10 min at room temperature to obtain a cell pellet; 100 μL RNase A solution was added to each sample, the cells were resuspended and then placed in a 37°C water bath for 30 min; 400 μL PI staining solution was added and mixed, and incubated at 4°C in the dark for 30 min; the red fluorescence at an excitation wavelength of 488 nm was recorded by flow cytometry.

[0125] 6. Senescence detection of hENFc matrix-enhanced hMSCs

[0126] For senescence-associated β-galactosidase assay, MSCs were detected using the SA-β-gal staining kit at passages 6, 11, and 15. T and MSC EN Briefly, cells were cultured on modified or non-modified 6-well plates until 80% confluence, washed with PBS and fixed with 4% formaldehyde for 15 min; then, cells were incubated with freshly prepared β-galactosidase staining solution in a CO2-free oven overnight at 37°C; after washing with PBS three times, photos were taken using an optical microscope, and the number of cells positively stained for β-galactosidase was counted for every 200 cells randomly selected in the field of view.

[0127] 7. Apoptosis detection of hENFc matrix-enhanced hMSCs

[0128] At passages 6, 11, and 15, 1 × 10 6 MSC T and MSC EN Cells; resuspend the cells with 1ml of Binding Buffer diluted with deionized water (diluted 1:9); add 5μL of AnnexinV / FITC to every 100μL of cell suspension and mix well, incubate at room temperature in the dark for 5min; add 5μL of propidium iodide solution and 400μL of PBS, place in a 5ml flow tube, and immediately perform flow cytometry detection. In addition, the unstained cells are used as blank tubes; the tubes without propidium iodide and only AnnexinV / FITC are used as single staining tubes.

[0129] 8. Detection of differentiation potential of hENFc matrix-enabled hMSCs

[0130] The sixth generation MSC T 11th generation MSC T and 11th generation MSC ENThe cells were cultured in differentiation medium for 14 days, and the tri-lineage differentiation ability of hMSCs was detected. For osteogenic differentiation: osteogenesis was induced by culturing cells in DF12 supplemented with 50 μg / mL ascorbic acid 2-phosphate, 10 nM dexamethasone (Dex), 10 mM β-glycerophosphate and 10% FBS, 1% penicillin-streptomycin, and mature cells after differentiation were stained with Alizarin Red S solution; for adipogenic differentiation: adipogenesis was induced by culturing cells in DF12 supplemented with 10% FBS, 50 μg / mL ascorbic acid 2-phosphate, 1 μM Dex, 0.1 mM indomethacin and 4 U / L insulin, and mature cells were detected using Oil Red O staining kit; for chondrogenic differentiation: chondrogenic differentiation was induced by culturing cells in DF12 supplemented with 2% FBS, 50 μg / mL ascorbic acid 2-phosphate, 50 μg / mL L-proline, 100 nm Dex, 10 ng / mL Chondrogenesis was induced by culturing cells in DF12 medium with TGF-β1, 1% human insulin, transferrin, and sodium selenite, and mature cells were detected using toluidine blue chondrogenic solution.

[0131] The following primers were used for PCR to detect the expression of genes related to the three-phase differentiation:

[0132]

[0133]

[0134] 9. Detection of tumorigenicity of hENFc matrix-enhanced hMSCs

[0135] To examine the tumorigenicity of hENFc-enabled hMSCs, 1×10 7 6th generation MSC T (n=3) and 11th generation MSC EN (n=6) The cells were subcutaneously transplanted into the abdomen of 4-week-old NOD-SCID mice; the weight of the mice was measured every 3 days and the volume of the tumor was measured according to the following formula. The mice were killed after seven weeks of continuous feeding. 7 Mice bearing human glioblastoma (U87) (n=3) served as positive controls, and mice injected with an equal volume of PBS (n=3) served as negative controls.

[0136]

[0137] 10. Results

[0138] 10.1 Effect of hENFc matrix on the proliferation of hMSCs expanded in vitro

[0139] Although the proliferation capacity of all cells gradually weakened with the passage of cells, the number of cells harvested in each passage also gradually decreased ( Figure 3However, hMSCs (MSC T ), the proliferation ability of hMSCs cultured in the cadherin fusion protein matrix was well maintained, especially hMSCs cultured in the hENFc matrix (MSC EN ), the cell doubling time remained within 25 hrs until the eleventh generation, and the proliferation capacity did not change significantly. This indicates that the hENFc matrix effectively maintains the in vitro proliferation activity of hMSCs through the cell-cell interaction mediated by biomimetic cell E-cadherin and N-cadherin. In addition, the size of hMSCs cultured in the hENFc matrix did not change with the passage expansion. EN The volume is always 2000μm 3 (diameter is about 16μm) Figure 4 ).

[0140] 9.2 Effect of hENFc matrix on β-catenin nuclear translocation of in vitro expanded hMSCs

[0141] like Figure 5 As shown in the figure, hMSCs cultured in vitro lost the expression of E-cadherin. Although the expression of N-cadherin was retained, it was gradually lost as the culture progressed. The use of biomimetic hENFc matrix culture restored the expression of endogenous E-cadherin in hMSCs and maintained the presence of N-cadherin. The expression of cadherin on the membrane plays an important role in the cadherin-catenin complex, and the combination of hE-cad-Fc and hN-cad-Fc matrix (at a mass ratio of 1:1) promoted the nuclear translocation of β-catenin ( Figure 6 ).

[0142] 9.3 Effect of hENFc matrix on the maintenance of stemness of in vitro expanded hMSCs

[0143] E-cadherin plays a key role in maintaining stem cell stemness. We continued to investigate the expression of hMSC stemness indicator OCT4 during long-term passage. Gene and protein detection results showed that OCT4 was expressed in hMSCs. T In comparison, MSC EN The stemness of MSCs was maintained in serial passages, and the sixth, eleventh and fifteenth passages EN The stemness is higher than that of MSC T . More importantly, the eleventh generation MSC EN The stemness is significantly better than that of the sixth generation MSCs T ( Figure 7 ).

[0144] 9.4 Effects of hENFc matrix on senescence and apoptosis of hMSCs expanded in vitro

[0145] Replicative senescence is the biggest obstacle to hMSCs culture in vitro, which leads to the loss of cell proliferation ability and changes in secretion profile, limiting the therapeutic efficacy of cells. Through cell cycle detection, we found that each generation of MSCs EN The number of cells in S phase was significantly higher than that of MSCs T β-galactosidase staining further confirmed that MSC EN The percentage of senescent cells in the MSCs was always below 10% and significantly less than that in the T ( Figure 8 ). The result is Fig. 9 As shown, after long-term in vitro culture, MSC T A large number of apoptosis occurred, and MSC EN The proportion of apoptotic cells did not change significantly with the increase in passage number, which made the eleventh generation MSCs EN The proportion of senescent and apoptotic cells in the sixth generation MSCs T Basically the same.

[0146] We further investigated the differentiation capacity of hMSCs at each generation and compared it with the eleventh generation MSCs. T (MSC T -P11) compared to the eleventh generation MSC EN (MSC EN -P11) and the sixth generation of MSC EN (MSC T -P6) found more significant Oil Red O-positive lipid droplets and PPAR-γ, CEBPA, and LPL gene expressions ( Fig.10 A), MSC EN -P11 and MSC T There was no statistical difference between P6 and P7. This proves that hENFc matrix can enhance the ability of cultured MSCs. EN -P11 and conventionally cultured MSCs T -P6 has the same adipogenic differentiation properties. EN -P11 cells were also detected in the cells after directed differentiation by alizarin red S staining and toluidine blue staining. Fluorescence quantitative PCR detection of the expression of mature bone and cartilage markers also confirmed that MSC EN -P11 and MSC T -P6 has the same osteogenic ( Fig.10 B) and cartilage ( Fig.10 C) differentiation potential.

[0147] 9.5 Safety analysis of hENFc matrix-enhanced hMSCs

[0148] The in vitro expansion and culture of MSCs inevitably leads to cell replicative senescence, which in turn leads to cell genomic instability, a serious defect that hinders the development of MSC-based cell therapy industry. Therefore, we examined the use of hENFc matrix to empower MSCs cultured to the eleventh generation. EN Compared with conventional culture to the sixth generation of MSC T The results of the analysis of the expression levels of genes related to genome stability transformation in transcriptome sequencing data showed that ( Fig.11 ), 11th generation MSC EN MSCs cultured normally at the sixth generation T There was no statistical difference in the expression of oncogenes and tumor suppressor genes between cells, and there was no significant change in the expression levels of c-Myc, P21, P53 and P16 genes. Karyotype analysis did not find clones with chromosomal abnormalities, and no chromosomal translocation, deletion or change in chromosome number was observed. hENFc-enabled hMSCs maintained chromosomal stability ( Fig.12 ).

[0149] Tumorigenicity is another risk factor that needs to be considered in the clinical application of MSCs. Compared with iPSCs and ESCs, although MSCs themselves do not have tumorigenicity, genomic instability and the possibility of forming teratomas are very low, in order to ensure the safety of hENFc matrix for long-term empowerment of hMSCs in clinical applications, we will use MSCs that meet clinical standards T hENFc empowers MSCs cultured to the eleventh generation EN and human astrocytic glioblastoma cells (U87) were injected subcutaneously into nude mice. Seven weeks later, the potential for these cells to form tumors was studied. Fig.13 As shown in A, mice inoculated with U87 developed soybean-sized lumps at the injection site, while mice inoculated with MSC T and MSC EN In order to monitor the overall health of the mice, we measured the body weight of the mice. The results showed that the nude mice inoculated with PBS, MSC T MSC EN There was no statistical difference in body weight between the mice of the U87 group and the mice of the Fig.13 B) Seeding MSCs T and MSC EN No bulge formed at the inoculation site of the mice. Fig.13 C) These results indicate that hMSCs cultured to the eleventh generation after hENFc empowerment are not tumorigenic and have no adverse effects on animals, which meets the clinical application standards of hMSCs.

[0150] Example 3: MSCEN Application in anti-inflammatory and immune regulation

[0151] 1. Experimental Methods

[0152] 1.1 Detection of inflammatory responsiveness of hENFc matrix-enabled hMSCs

[0153] MSCT and MSCEN were both measured at 1×10 per square centimeter. 4 The cells were inoculated at a density of 100 cells / mL on a 96-well TCPS plate and cultured for 24 hours; then IFN-γ was added to the culture medium at final concentrations of 0, 5, 10, 15, 20, 25, and 30 ng / mL and cultured for 24 hours;

[0154] Cell viability was detected as follows: MSCs were seeded into 96-well TCPS, hEFc, hNFc or hENFc matrix, incubated for 4, 24, and 48 hours, the supernatant was removed, and the cells were washed 3 times with PBS. 100 μL of CCK-8 (1:10 dilution) reagent was added to each well, incubated at 37°C for 4 hours, and the absorbance was detected at 450 nm using an ELISA reader.

[0155] MSCT and MSCEN were both measured at 1×10 per square centimeter. 4 The cells were inoculated into 6-well TCPS plates at a density of 100 cells / mL and cultured for 24 hours; IFN-γ with a final concentration of 10 ng / mL was then added to the culture medium and cultured for 24 hours; the supernatant and cells were then collected for subsequent detection.

[0156] The content of soluble tumor necrosis factor receptor 1 (sTNFR1) in the supernatant was detected by Elisa method. First, all supernatants were centrifuged at 300×g for 10 minutes to remove the precipitate; 300μL of diluted washing solution (1:20 dilution) was added to soak the plate for 30 seconds; all washing solutions were discarded and patted dry on absorbent paper, and 100μL of culture medium or cell culture supernatant sample or diluted standard was added to each well; 50μL of detection antibody (1:100 dilution) was added to each well; incubated at room temperature for 2hrs; the liquid was discarded, and 300μL of washing solution was added to each well to wash the plate 6 times. After each wash, the washing solution was discarded and the plate was patted dry; 100μL of Horseradish peroxidase-labeled streptavidin (1:100 dilution), incubate at room temperature for 45 minutes; discard the liquid, add 300 μL of washing solution to each well and wash the plate 6 times, discard the washing solution after each wash and pat the plate dry; add 100 μL of chromogenic substrate TMB to each well, incubate at room temperature in the dark for 30 minutes; add 100 μL of stop solution to each well, at which time the color changes from blue to yellow; within 30 minutes, use an ELISA reader for dual-wavelength detection, measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 570nm and calculate the concentration. 1.2MSC ENCo-culture experiment with peripheral blood mononuclear cells

[0157] PBMCs were isolated from whole blood donated by healthy donors, and these lipid layers were obtained by Histopaque density gradient centrifugation (ρ=1.077g / cm3); MSCT and MSCEN were then inoculated into 96-well plates and cultured for 24 hours using DF12 medium containing 10% FBS supplemented with mitomycin C; DF12 medium was replaced with RPMI-1640 medium supplemented with 10% FBS, and PBMCs were added to hMSCs at a ratio of 10:1; 1μg / mL of anti-human CD3 and anti-human CD28 monoclonal antibodies were added to activate PBMCs; after 24 hours of co-culture, all the culture medium was aspirated and centrifuged at 2000rpm for 10min, and the supernatant and cell pellet of the co-culture were collected for further detection; PBMCs cultured alone were used as negative controls, and each group of experiments was repeated 6 times.

[0158] For the PBMC proliferation test, the separated PBMC suspension was centrifuged at 300×g for 10 min, and all the supernatant was discarded; 1 ml of PBS containing 0.1% FBS was added to resuspend the cells, and 2 μM CFSE was added. After thorough mixing, the cells were cultured at 37°C, 5% CO2 for 10 min; 3 volumes of pre-cooled RPMI1640 culture medium were added and the culture was terminated at 4°C for 5 min; centrifuged at 300×g for 10 min, the supernatant was discarded, 1 ml of PBS containing 0.1% FBS was added to resuspend the cells, and the cells were centrifuged at 300×g for 10 min, the supernatant was discarded, and the co-culture experiment was then carried out with the obtained cells; after the co-culture was completed, the cells in the culture medium were collected and analyzed by flow cytometer.

[0159] For PBMC apoptosis detection, the following steps were performed: 1 × 10 cells were harvested at passage 6, 11, and 15 using EDTA-free trypsin. 6 MSC T and MSC EN Cells; resuspend the cells with 1ml of Binding Buffer diluted with deionized water (diluted 1:9); add 5μL of AnnexinV / FITC to every 100μL of cell suspension and mix well, incubate at room temperature in the dark for 5min; add 5μL of propidium iodide solution and 400μL of PBS, place in a 5ml flow tube, and immediately perform flow cytometry detection. In addition, unstained cells are used as blank tubes; the tubes without propidium iodide and only AnnexinV / FITC are used as single-stained tubes.

[0160] For the determination of T lymphocytes and T lymphocyte subsets in PBMC cells, 1 ml of PBS was added to the cell pellet obtained after the above co-culture to resuspend the cells, centrifuged at 300 × g for 10 min, the supernatant was discarded, and the following antibodies were used to characterize PBMC subsets: CD4+T cells (FITC-labeled anti-human CD4 antibody, diluted 1:500), CD8+T cells (FITC-labeled anti-human CD8 antibody, diluted 1:500), Th1 / Th2 (FITC-labeled anti-human CD4, PE-labeled anti-human IL4, APC-labeled anti-human IFN-γ antibodies, diluted 1:500), Treg (FITC-labeled anti-human CD4, PE-labeled anti-human F After adding the antibodies, incubate at 4°C in the dark for 30 min. Note that for Foxp3 staining, first add 750 μL of membrane permeabilization agent and incubate at 4°C in the dark for 60 min. Add 1 ml of PBS to wash the cells once, centrifuge at 300 × g for 10 min, discard the supernatant, add 500 μL of PBS to resuspend, filter and analyze the cells by flow cytometry.

[0161] For the detection of the secretion of TNF-α, IFN-γ, IL-1β and TGF-β factors in the co-culture system, the supernatant obtained after the above co-culture was analyzed according to the above Elisa experiment.

[0162] 2. Results

[0163] 2.1 Effect of hENFc matrix empowerment on the responsiveness of hMSCs to inflammatory environment

[0164] The increase of inflammatory mediators can affect the activity of MSCs and the expression of immune regulatory factors. In order to characterize the response of hMSCs to inflammatory environment stimulation, we stimulated MSCs with gradient concentrations of IFN-γ (0-30ng / mL). T and MSC EN After 24 hrs, cell viability and factor expression were characterized. Fig.14 The results showed that with the increase of IFN-γ concentration, the cell viability of hMSCs decreased significantly. When the concentration of IFN-γ was greater than 15 ng / mL, MSC EN The activity was significantly higher than that of MSC T IFN-γ stimulation can upregulate the expression of pro-apoptotic protein Bax and inhibit the expression of anti-apoptotic protein Bcl-2, further activating Caspase3 to execute cell apoptosis, but MSC EN The anti-apoptosis ability is significantly better than that of MSCs TIn a non-inflammatory environment, hMSCs basically do not express the anti-inflammatory factor IDO, but after IFN-γ stimulation, they immediately express IDO at a high level. EN The MSCs have stronger responsiveness, with the gene and protein expression of IDO being twice that of MSCTs. EN Has a stronger release capacity of sTNFR1 ( Fig.15 ). These results indicate that hMSCs empowered by hENFc matrix not only have better survival ability in the lesion microenvironment, but also have stronger environmental perception and anti-inflammatory ability. The IDO primer sequence is as follows:

[0165]

[0166] 2.2 MSC EN Effects on proliferation and differentiation of peripheral blood monocytes

[0167] In immunology, immune responses are widely evaluated by the response of PBMCs. Based on the ability of MSCs to affect PBMC cell proliferation, apoptosis, and differentiation in co-culture systems, we evaluated MSCs. EN Flow cytometry analysis showed that compared with untreated and MSC T In comparison, MSC EN Can significantly inhibit PBMC proliferation ( Fig.16 A) and promote cell apoptosis ( Fig.16 B), MSC EN It also inhibited the proportion of CD4+ and CD8+ T cell subsets ( Fig.16 C and Fig.16 D)

[0168] It is generally believed in immunology that initial CD4+T cells differentiate into different cell subtypes after activation, namely, type 1 T helper cells (Th1), type 2 T helper cells (Th2), interleukin-17-producing T helper cells (Th17) or regulatory T cells (Treg) to coordinate various immune responses. We found that MSCs reduced the proportion of Th1 (CD4+IFN-γ+) and Th17 (CD4+IL17A+) ( Fig.17 A and Fig.17 C), increased the proportion of Th2 (CD4+IL4+) and Treg (CD4+CD25+FoxP3+) ( Fig.17 B and Fig.17 D) Compared with MSCT, MSCEN has a more significant effect on T cell differentiation, further promoting the Th1 / Th2 balance to tilt toward Th2 cells and the Th17 / Treg balance to tilt toward Treg cells.

[0169] like Fig.18 As shown in Figure 2, in the MSC-PBMC co-culture group, the levels of pro-inflammatory factors TNF-α, IFN-γ, and IL-1β were reduced, and the level of anti-inflammatory factor TGF-β was increased. EN The changes in IFN-γ and IL-1β levels in the co-culture system were greater. It is generally believed that immune balance, especially the balance between pro-inflammatory factors and immunosuppressive cytokines, is crucial to maintaining the balance of the human body. These results indicate that the empowered culture of hENFc can improve the efficacy of hMSCs in inflammatory and autoimmune diseases.

[0170] Example 4: MSC EN Treating systemic lupus erythematosus

[0171] 1. Experimental Methods

[0172] 1.1 Modeling of systemic lupus erythematosus nephritis

[0173] The systemic lupus erythematosus (SLE) model provides a valuable model for evaluating the immunomodulatory and therapeutic potential of MSCs due to its autoimmune nature, multi-organ involvement, and pathological similarities with human SLE. We established a SLE model using MRL / lpr mice ( Fig.19 At the end of the two-week transplantation period, transplanted MSCs were observed EN The lupus symptoms of mice in the treatment group were significantly improved, compared with the PBS treatment group and the MSC T The mice in the treatment group were in sharp contrast.

[0174] 1.2MSC EN transplant

[0175] Eighteen 8-week-old female MRL / lpr lupus mice were housed in an SPF environment for 2 weeks. The treatment group was injected with 1×10 6 MSC T MSC EN The control group was injected with 200 μL of saline; the animals were weighed weekly; all animal husbandry and experiments strictly followed the animal care and use guidelines approved by Nanjing University Drum Tower Hospital, and appropriate measures were taken to ensure that the animals had minimal pain and discomfort. In the 12th week, blood was collected from the eyeballs after intraperitoneal anesthesia, and the animals were killed by cervical dislocation. The spleen and lymph nodes were weighed, and some kidneys were immediately placed in liquid nitrogen for storage.

[0176] Part of the kidney was fixed in formaldehyde overnight, and the specimens were treated with 70% ethanol, 80% ethanol, 90% ethanol, and 95% ethanol in order from low concentration to high concentration for 2 hours, and dehydrated with anhydrous ethanol for 2 hours, which was repeated once. The tissue block was then placed in xylene to make it transparent, and the transparent sample was placed in liquid paraffin. After the paraffin was completely immersed in the tissue block, it was embedded and cooled to fix into a block; the embedded sample was sliced ​​on a paraffin slicer, and then placed in warm water for unfolding. The unfolded paraffin slice was carefully attached to a glass slide, and then the paraffin patch was dried at a constant temperature of 45°C and stored at room temperature.

[0177] Part of the kidney was fixed in formaldehyde overnight and dehydrated with 30% sucrose solution until the kidney sank to the bottom; the tissue was removed, washed twice with pre-cooled PBS, and then an appropriate amount of OCT embedding medium was added to immerse the tissue; the pre-cooled OCT was fixed on a slicer, cut into thin slices, attached to a slide, and stored in a -80°C refrigerator for later use.

[0178] 1.3 Mouse urine protein detection

[0179] Use a metabolic cage to collect mouse urine, dilute the urine of the mouse to be tested to an appropriate concentration, dilute the total sample volume to 5 μL, add 195 μL of Coomassie Brilliant Blue G250 solution, mix thoroughly, and let stand for 2 minutes; measure the absorbance at 595 nm, and calculate the urine protein concentration.

[0180] 1.4 Detection of mouse serum ds-DNA and TNF-α

[0181] After blood was collected from the medial canthus vein of the mouse, it was placed in a 1.5 mL centrifuge tube and centrifuged at 300 × g for 10 min. The supernatant was transferred to a new 1.5 mL centrifuge tube and allowed to stand for 30 min. The supernatant was serum, which was transferred to a new centrifuge tube and stored in a -80 °C refrigerator for later use. Elisa assay was used to analyze the content of ds-DNA and TNF-α in mouse serum.

[0182] 1.5 Mouse kidney pathology staining

[0183] The paraffin sections were dewaxed and hydrated by treating with xylene for 20 min, anhydrous ethanol for 3 min, 95% ethanol for 3 min, 95% ethanol for 3 min, 90% ethanol for 3 min, 80% ethanol for 3 min, and 70% ethanol for 3 min.

[0184] ① For H&E staining: Place the processed sections in a hematoxylin staining box and stain for 5 minutes; wash off the floating color in water and rinse gently with running water for 10 minutes; treat with hydrochloric acid and alcohol for 1 second; wash off the floating color in water and rinse with running water for 8 minutes; stain with eosin for 90 seconds; wash off the floating color in water and rinse gently with running water for 10 minutes.

[0185] ② For PAS staining: Place the processed sections in periodic acid solution and oxidize at room temperature for 25 minutes; rinse twice with running water for 2 minutes each time, and gently rinse twice with distilled water for 2 minutes each time; stain with Schiff Reagent at 37℃ in the dark for 10 minutes; wash twice with sodium sulfite solution for 2 minutes each time; rinse twice with running water for 2 minutes each time, and gently rinse twice with distilled water for 2 minutes each time; stain in a hematoxylin staining box for 5 minutes; wash off floating color in water, and gently rinse with running water for 10 minutes.

[0186] ③ For Masson staining: Place the processed sections in Weigert iron hematoxylin staining solution for 10 minutes; rinse with running water for 10 minutes; return to blue with Masson bluing solution for 5 minutes; rinse with running water for 10 minutes, rinse with distilled water twice, 2 minutes each time; stain with Ponceau fuchsin staining solution for 10 minutes; wash with weak acid working solution (distilled water: weak acid solution = 2:1) for 1 minute; wash with 1% phosphomolybdic acid solution for 2 minutes; wash with weak acid working solution for 1 minute; directly place in aniline blue staining solution for 2 minutes; wash with weak acid working solution for 1 minute.

[0187] After all samples were stained, they were dehydrated by treating with 70% alcohol for 1 sec, 80% alcohol for 1 sec, 85% alcohol for 1 sec, 90% alcohol for 1 sec, 95% alcohol for 30 sec, and anhydrous ethanol for 30 sec; dried in a ventilated place, immersed in xylene for 5 min; immersed in fresh xylene for 5 min; air-dried in a fume hood, sealed with neutral resin, and stored at room temperature.

[0188] 1.6 Mouse kidney tissue pathological scoring

[0189] Referring to the study by Tao et al., renal pathology scores were scored based on the H&E, PAS, and Masson staining results of the sections according to the degree of inflammatory cell infiltration and mesangial proliferation in the glomeruli, renal interstitium, blood vessels, etc.:

[0190] Table 3.3 Renal pathology scores

[0191]

[0192] 1.7 Immunofluorescence staining of mouse kidney

[0193] The frozen sections were rinsed in running water for 5 min to wash off the OCT, and immunofluorescence staining was performed to detect the expression and distribution of IgG and C3 in different kidney tissues.

[0194] 1.8 Preparation of single cell suspension from mouse perirenal lymph nodes

[0195] Disinfect the abdomen with alcohol, remove the spleen or perirenal lymph nodes, place them in a culture dish covered with PBS, grind the perirenal lymph nodes, add a filter to grind, and filter; add 5mL of red blood cell lysis buffer, resuspend and incubate at room temperature for 10min; add 5mL of PBS containing 2% FBS, centrifuge at 1500rpm for 5min; remove the supernatant, add 5mL of PBS containing 2% FBS to filter; resuspend with 10mL of PBS containing 2% FBS; take a 96-well flat-bottom plate, add 10μL of the suspension and add 90μL of PBS, take another 10μL of it and add 90μL of PBS, and then count.

[0196] 1.9 Detection of T cell subsets in mouse spleen and perirenal lymph nodes

[0197] For T cell proliferation and differentiation staining, 1×10 6 The cells were placed in a 96-well U-shaped plate, 5 μL countbeads were added to each well, and the plate was centrifuged at 2000 rpm for 5 min; anti-mouse TCRβ antibody labeled with BV421 (1:500 dilution), anti-mouse CD4 antibody labeled with FITC (1:500 dilution), anti-mouse CD8 antibody labeled with BV510 (1:500 dilution), anti-mouse CD62L antibody labeled with BV605 (1:500 dilution), anti-mouse CD44 antibody labeled with BV711 (1:500 dilution), anti-mouse CD25 antibody labeled with APC (1:500 dilution), anti-mouse CD69 antibody labeled with Percp Cy5.5 (1:500 dilution), anti-mouse Ki67 antibody labeled with PE Cy7 (1:500 dilution), anti-mouse Foxp3 antibody labeled with PE (1:500 dilution), and anti-mouse live Dye antibody (1:500 dilution) was added and incubated at 4℃ in the dark for 30min. Note that for nuclear antibody staining, 750μL of membrane permeabilization agent was added first and incubated at 4℃ in the dark for 60min; 1ml of PBS containing 2% FBS was added to wash the cells once, centrifuged at 300×g for 10min, and the supernatant was discarded; resuspended with 150μL PBS containing 2% FBS, filtered and analyzed by flow cytometry.

[0198] For T subpopulation staining, 2 × 10 510 cells were placed in a 96-well U-shaped plate, and 200uL of 1640 complete medium containing PMA, INO and BFA (1:1000 dilution) was added to each well. The cells were resuspended after mixing and cultured at 37°C for 6h; centrifuged at 2000rpm for 5min; e780-labeled anti-mouse live cell antibody (1:500 dilution), FITC-labeled anti-mouse CD4 antibody (1:500 dilution), BV510-labeled anti-mouse CD8 antibody (1:500 dilution), BV421-labeled anti-mouse IL17 antibody (1:500 dilution), APC After adding 1 mL of PBS containing 2% FBS, wash the cells once, centrifuge at 300 × g for 10 min, and discard the supernatant; resuspend with 150 μL PBS containing 2% FBS, filter and analyze the cells by flow cytometry.

[0199] 2. Results

[0200] 2.1 Detection of physiological and biochemical indicators of mice in different treatment groups

[0201] Various parameters such as weight loss, increased proteinuria, increased anti-dsDNA antibody levels, and TNF-α expression are the most commonly used indicators for clinical diagnosis of SLE. In order to study the disease status of MRL / lpr mice after MSC transplantation, the weight of mice, the protein content in urine, and the concentration of anti-ds-DNA antibodies and TNF-α in the blood of mice were detected by Elisa kits. It was found that there was no significant difference between the PBS treatment group and the MSC group. T Compared with the treated group, MSC EN The physiological and biochemical indicators of mice in the treatment group were more normal ( Fig. 20 ).

[0202] 2.2 MSC EN Effects of transplantation on renal tissue pathology and antibody deposition

[0203] We next tested MSC EN We investigated whether in vivo treatment might have beneficial effects on renal histopathology and autoantibodies in SLE mice. The results of renal histopathology of MRL / lpr mice observed and analyzed using H&E, PAS, and Masson pathology staining showed that hMSCs-treated mice showed reversal of glomerular damage. In addition, Masson staining as well as H&E and PAS staining also showed that hMSCs-treated mice showed reversal of glomerular damage compared with the PBS-treated group and MSCs-treated mice. T Compared with the treatment group, MSC ENTreated mice also showed significant reductions in glomerular, interstitial, and perivascular damage ( Fig.21 ). It is worth noting that compared with the PBS treatment group and MSC T Compared with the treatment group, MSC EN The thickening of glomerular mesangium and basement membrane, inflammatory cell infiltration, interstitial fibrosis and occasional crescent formation were significantly reduced in the treated mice ( Fig. 22 ).

[0204] Antibody deposition plays a key role in the pathogenesis of LN. Immunofluorescence results showed that transplanted MSCs EN The deposition of C3 and IgG antibodies in the kidneys of MRL / lpr mice was significantly lower than that in the PBS-treated group and MSC-treated group. T Treatment group ( Fig.23 These results indicate that MSC EN In vivo treatment of MRL / lpr mice ameliorated the lupus-like phenotype.

[0205] 2.3 Analysis of CD4+ and CD8+ cell status in perinephric lymph nodes and spleen of mice in different treatment groups

[0206] The results are as follows Fig.24 Display, MSC EN Treatment with MSCs reduced the number of CD4+ and CD8+ T cells in the perinephric lymph nodes, inhibited their activation (CD69+), proliferation (Ki67+), and effector function (CD44+CD62L-), and also increased the proportion of unsensitized T cells (CD44-CD62L+). We continued to analyze the proportion of Th1 cells, Th2 cells, and Treg cells in the perinephric lymph nodes of mice to determine whether MSCs EN Effects of treatment on various T helper cell subsets. Fig.25 The results showed that hMSCs treatment resulted in a decrease in the proportion of CD4+IFN-γ+ cells (Th1) and an increase in CD4+IL4+ cells (Th2) and CD4+IFN-γ+ cells CD4+CD25+Foxp3+ cells (Treg). EN Compared with the PBS treatment group and MSC T Compared with the treatment group, MSC EN The Th1 / Th2 ratio in the renal lymph nodes of treated mice also decreased, indicating that MSC EN Th2 cells predominated in the perinephric lymph nodes of treated mice.

[0207] We also observed similar results in the spleen ( Fig.26 and Fig. 27 These findings are consistent with MSC EN Than MSC TThese results demonstrate that MSCs have a stronger ability to regulate T cell subsets and promote an anti-inflammatory environment. EN The important role played by induced T cell responses and Th cell subset balance in LN highlights the advantages of hENFc matrix-enabled culture in improving the therapeutic effect of hMSCs.

[0208] Example 5: MSC EN Treatment of diabetic nephropathy in rats

[0209] 1. Experimental Methods

[0210] 1.1 Establishment of rat model of type 2 diabetic nephropathy

[0211] All 30 SD rats were adaptively fed for 1 week, and then fed with high-fat diet (n=20) and normal diet (n=10) for 8 consecutive weeks to ensure that the rats had normal blood sugar (blood sugar <250 mg / dL); after 8 weeks, all rats were fasted for 12 hours, and rats fed with high-fat diet were intraperitoneally injected with STZ dissolved in sodium citrate solution at 35 mg / kg, while rats fed with normal diet were intraperitoneally injected with the same amount of sodium citrate solution. Blood sugar was continuously tested for 3 days. When the blood sugar concentration of rats in the high-fat diet plus STZ injection group was ≥250 mg / dL, it indicated that the type 2 diabetes model was successfully established ( Fig.28 ).

[0212] After the type 2 diabetes model was successfully established, the rats were gradually fed a normal diet, the rats were observed and their blood and urine indicators were tested; after 2 weeks of continuous culture, the rats' urine was collected for 24 hours. When the urine protein was >20mg / 24hrs and significantly higher than that of the normal control group, it was considered that renal damage occurred at this time, and the diabetic nephropathy (DKD) rat model was successfully established. All animal husbandry and experiments strictly followed the animal care and use guidelines approved by Nanjing University Drum Tower Hospital, and appropriate measures were taken to ensure that the animals had minimal pain and discomfort.

[0213] 1.2 MSC EN transplant

[0214] After the diabetic nephropathy model was successfully established, the rats were randomly divided into the disease group injected with normal saline (abbreviated as DKD), the disease group injected with MSC T The treatment group (abbreviated as KDK+MSC T ) and injection of MSC EN The treatment group (abbreviated as KDK+MSC T ), and normal rats without modeling were used as control group (Sham), with at least 5 rats in each group. The treatment group was injected with 2×106 MSCs through the tail vein at the 11th and 12th week, and the control group was injected with 500μL of normal saline.

[0215] 1.3 Collection of blood, urine and tissue specimens from rats

[0216] Rats were weighed weekly. Blood was collected from the end of the tail vein to measure blood glucose. One day before the end of the experiment, urine and feces of rats were collected in a metabolic cage and frozen in a -80°C refrigerator. After two weeks of continuous treatment, the weight of the rats was first recorded, and then the rats were anesthetized, the chest cavity of the rats was opened, and the heart was punctured at the left atrial appendage with a 5mL syringe to collect blood and placed in an EP tube. Centrifuged at 300×g for 10 minutes, the upper liquid was aspirated, and the supernatant was aspirated as serum after standing for 30 minutes, and stored in a -80°C refrigerator for later use. After that, the abdominal cavity of the rats was opened, and the kidneys and spleen were taken out for weighing. Some kidneys were immediately placed in liquid nitrogen for storage; some kidneys were placed in formaldehyde for fixation overnight, and the specimens were treated with 70% ethanol, 80% ethanol, 90% ethanol, and 95% ethanol in order from low concentration to high concentration for 2 hours, dehydrated with anhydrous ethanol twice, each time for 1 hour, and then the tissue block was placed in xylene for transparency, and the transparent tissue was placed in molten paraffin. After the paraffin was completely immersed in the tissue block, it was embedded, cooled and fixed into blocks, and the pre-cooled wax blocks were fixed on the microtome, cut into thin slices, put in heated water to flatten, attached to the slide, dried in a 45℃ constant temperature box, and stored at room temperature; some kidneys were placed in formaldehyde for fixation overnight, dehydrated with 30% sucrose solution until the kidney sank to the bottom, the tissue was removed, washed twice with pre-cooled PBS, and an appropriate amount of OCT embedding agent was added to immerse the tissue, the pre-cooled OCT was fixed on the microtome, cut into thin slices, attached to the slide, and stored in a -80℃ refrigerator for later use.

[0217] 1.4 Biochemical index detection of rats

[0218] ① The renal index is calculated as follows:

[0219]

[0220] ② The spleen index is calculated as follows:

[0221]

[0222] ③Urine indicators:

[0223] After the rat urine was equilibrated to room temperature, it was centrifuged at 1000×g for 10 min and the supernatant was taken; the protein content in the rat urine was detected.

[0224] ④ Kidney indicators:

[0225] Take part of the kidney tissue, add 0.9% physiological saline, and homogenize the tissue at low speed with a tissue homogenizer on ice to make a 10% tissue homogenate; centrifuge at 8000×g and 4°C for 10 min, and take the supernatant for later use.

[0226] For malondialdehyde (MDA) detection: add 300 μL of MDA detection working solution to a 1.5 mL centrifuge tube; then add 100 μL of tissue homogenate supernatant to the assay tube and 100 μL of distilled water to the blank tube; add 100 μL of reagent three to all centrifuge tubes; after mixing, keep warm in a 100°C water bath for 60 min and then cool in an ice bath; centrifuge at 1000×g at room temperature for 10 min; aspirate 200 μL of supernatant in a 96-well plate and measure the absorbance of the sample at 532 nm and 600 nm.

[0227] For superoxide dismutase (SOD) detection, add the required reagents to the 96-well plate according to Table 3.4; after thorough mixing, incubate in a 37°C water bath for 30 minutes, and measure the absorbance of the sample at 450 nm.

[0228] Table 3.4 Renal pathology scoring table

[0229]

[0230] 1.5 Rat kidney pathological staining

[0231] After the prepared paraffin sections were dewaxed and hydrated, the rat kidneys were subjected to pathological staining according to the H&E, PAS and Masson staining methods of Example 3.

[0232] 1.6 Immunofluorescence staining of rat kidney

[0233] The prepared frozen sections were rinsed with running water for 5 minutes to wash off the OCT, and immunofluorescence staining was performed according to the method of Example 3 to detect the expression and distribution of synaptopodin in different kidney tissues.

[0234] 1.7 Immunohistochemical staining of rat kidney

[0235] The prepared frozen sections were rinsed with running water for 5 minutes to wash off the OCT; the sections were placed in a wet box, 100 μL of 3% hydrogen peroxide was added, and incubated at room temperature for 10 minutes; PBS was washed 3 times, 2 minutes each time, and distilled water was washed for 3 minutes; the water was dried, and 100 μL of goat serum was added to block at room temperature for 10 minutes; the blocking solution was discarded, and 100 μL of rabbit anti-NRF2 (1:1000 dilution) antibody was added and incubated at 4°C overnight; PBS was washed 3 times, 3 minutes each time, and distilled water was washed for 3 minutes; the water was dried, and 100 μL of Incubate with HRP-labeled goat anti-rabbit IgG (H+L) antibody (1:1000 dilution) at room temperature for 2 hrs; wash with PBS 3 times, 3 min each time, and distilled water for 3 min; shake dry, add 100 μL streptavidin-peroxidase solution, and incubate at room temperature for 10 min; wash with PBS 3 times, 3 min each time, and distilled water for 3 min; add 100 μL DAB solution, and observe under a microscope for 3-10 min until staining is appropriate; rinse with tap water and then stain with hematoxylin for 2 min, and rinse with tap water for blueing; differentiate with 1% hydrochloric acid alcohol for 3 sec, and rinse with tap water for 3 min; treat with 70% alcohol for 1 sec, 80% alcohol for 1 sec, 85% alcohol for 1 s, 90% alcohol for 1 sec, 95% alcohol for 30 sec, and dehydrate with anhydrous ethanol for 30 sec; dry in a ventilated place, soak in xylene for 5 min, put in a ventilated kitchen to air dry, seal with neutral resin, and store at room temperature.

[0236] 1.8 Western blotting of rat kidney

[0237] First, weigh 50 mg of kidney tissue and place it in a 1.5 mL centrifuge tube, add 0.5 mL of protein lysis buffer; homogenize with a tissue homogenizer in an ice bath; then centrifuge at 4°C and 12000 rpm for 15 min, take the supernatant and measure the protein concentration and Western Blotting experiment according to the method in 2.3.7.2, and detect the protein levels of NRF2, HO-1, BCL-2, BAX, Nephrin and WT1 expressions in the kidneys of different treatments.

[0238] 2. Results

[0239] 2.1MSC EN Treatment of diabetic nephropathy in rats

[0240] We established a rat DKD model by high glucose feeding and STZ injection after one week of adaptive feeding and evaluated the MSC EN At the end of the two-week transplantation period, the MSCs in the untreated group T Compared with the rats in the treated group, MSC ENThe rats in the treatment group showed significant improvement in urine, serum and other related diabetes symptoms, alleviated kidney tissue pathological symptoms, and the progression of diabetic nephropathy was significantly delayed.

[0241] 2.2MSC EN Effects of transplantation on physiological and biochemical parameters in type 2 diabetic rats

[0242] During DKD modeling and hMSCs intervention, we observed that DKD rats significantly increased their feed and water intake compared with the control group. Compared with DKD rats, rats in the hMSCs-treated group reduced their water intake ( Fig.29 A) and feed quantity ( Fig.29 B). Two weeks after hMSCs transplantation, the body weight of DKD rats decreased significantly ( Fig.29 C), blood sugar concentration slightly improved ( Fig.29 D). The 24-hour urine protein content, serum urea nitrogen (BUN) and serum creatinine (SCr) of DKD rats were significantly higher than those of the control group, while hMSCs treatment significantly inhibited this increase ( Fig.29 E. Fig.29 F and Fig.29 G) More importantly, with MSC T The therapeutic ratio of MSC EN The treatment significantly inhibited the increase of blood glucose, 24-hour urine protein, BUN and SCr in rats.

[0243] 2.3MSC EN Effects of transplantation on renal morphology in type 2 diabetic rats

[0244] Compared with DKD group and MSC T Compared with the treatment group, MSC EN The kidney weight and kidney weight index of the rats in the treatment group were significantly reduced ( Fig.30 ). H&E and PAS staining results showed that compared with the Sham group, the glomerular hypertrophy and mesangial matrix proliferation were obvious in the DKD group. T Compared with the treatment group, MSC EN The glomerular volume of the rats in the treatment group was basically normal, and the mesangial matrix was significantly reduced. Masson staining results showed that compared with the Sham group, the collagen deposition in the glomerular mesangial area of ​​the rats in the DKD group was increased, while MSC EN Transplantation effectively reduces glomerular fibrosis ( Fig.31 These results suggest that MSC EN It effectively improved the renal trauma of DKD rats and had a stronger protective effect on the kidneys of DKD rats.

[0245] 2.4MSC EN Effects of transplantation on renal oxidative damage in type 2 diabetic rats

[0246] Oxidative damage caused by high glucose and hyperlipidemia in diabetic patients is one of the important reasons for the development of DKD. Malondialdehyde (MDA) is a product of lipid peroxidation, and superoxide dismutase (SOD) is one of the main antioxidant enzymes in cells. In DKD patients, due to the increased level of oxidative stress, lipid peroxidation is aggravated, and the activity of SOD is often affected. T Compared with the rats in the treatment group, the MDA content in the kidneys of the rats in the MSCEN treatment group was significantly decreased, and the SOD activity was enhanced ( Fig.32 ), effectively reducing cell damage caused by oxidative stress and alleviating the development of diabetic nephropathy.

[0247] Effect of 2.5hMSCs transplantation on renal apoptosis in type 2 diabetic rats

[0248] like Fig.33 As shown in Figure 2, in DKD rats, the activity of NRF2 is often inhibited, leading to increased levels of intracellular oxidative stress. HO-1 is an important antioxidant enzyme regulated by NRF2, which can produce a series of antioxidant, anti-inflammatory and anti-apoptotic products. In DKD rats, the expression of HO-1 is inhibited due to increased oxidative stress. Transplantation of hMSCs can effectively restore the expression of NRF2, further promote the expression of HO-1, reduce the BAX / Bcl-2 ratio, and reduce cell apoptosis in the kidney, thereby alleviating renal damage ( Fig.34 ). T Compared with the rats in the treated group, MSC EN The BAX / Bcl-2 ratio was lower in the treatment group, demonstrating that hENFc empowered hMSCs with stronger anti-apoptosis ability.

[0249] 2.6MSC EN Effects of transplantation on systemic inflammation in type 2 diabetic rats

[0250] Under hyperglycemia, the inflammatory response in diabetic tissues is activated, leading to increased infiltration of inflammatory cells and release of inflammatory factors. These inflammatory responses further aggravate kidney tissue damage and accelerate the development of diabetic nephropathy. To investigate whether hMSCs transplantation regulates the inflammatory response in DKD rats, we observed the spleen images of rats ( Fig.35 B) and its weight ( Fig.35 A) and spleen index ( Fig.35 C), the results showed that MSC EN The organ size and organ index of the spleen in the treated group were significantly lower than those in the other three groups. These results indicate that MSC EN The transplantation of MSCs has an inhibitory effect on spleen enlargement, which may be related to ENIt is closely related to the inhibition of lymphocyte proliferation. We continued to measure the expression of proinflammatory cytokines in the kidneys of rats, and the results showed that compared with the DKD group, the mRNA levels of IL-1β and TNF-α in the kidneys of rats in the hMSCs treatment group were reduced. The cell damage and inflammatory factor release caused by the inflammatory response induce the activation of fibroblasts, leading to renal interstitial fibrosis. Our results showed that compared with the MSCT treatment group, the expression of IL-1β and TNF-α in the kidneys of rats in the MSCEN treatment group was reduced, and the expression of TGF-β was further reduced ( Fig.35 D) Also at MSC EN A decrease in the concentrations of IL-1β, TNF-α, and TGF-β was observed in the serum of rats in the treatment group ( Fig.35 E) These results suggest that MSC EN It can effectively reduce the inflammatory response in the systemic and renal tissues of DKD rats, thereby delaying the progression of diabetic nephropathy.

[0251] 2.7MSC EN Effect of transplantation on renal function in rats with type 2 diabetes

[0252] Synaptopodin is related to maintaining the structure and function of the glomerular filtration membrane. It is involved in regulating the morphology and dynamic structure of podocytes and is essential for maintaining the filtration function of the glomerulus. Uremia-associated protein (Nephrin) is closely related to the selective permeability of the glomerular filtration membrane. It constructs the interstitial membrane of podocytes by interacting with other proteins, maintains the structural integrity of the filtration membrane, and participates in regulating glomerular blood filtration and urine formation. In the glomerulus, the expression of Wilms tumor gene 1 (WT1) is related to the formation of glomerular blasts and the differentiation of podocytes, while in the adult kidney, the expression of WT1 is related to maintaining the function of podocytes and regulating the function of renal tubules and interstitial cells. Through immunofluorescence staining and Western Blotting, we can find that compared with the treatment of MSCT, MSC EN Treatment with α-synaptopodin restored the expression and distribution of synaptopodin in the kidney of rats ( Fig.36 A), increased the expression of Nephrin and WT1 proteins ( Fig.36 B) Proved that MSC EN Transplantation plays an important role in repairing the glomerular filtration function, maintaining the integrity of the filtration membrane, and regulating the glomerular structure and function of DKD rats.

[0253] Example 6: MSC EN Treating systemic sclerosis

[0254] 1. Experimental Methods

[0255] 1.1 Systemic sclerosis mouse model establishment and treatment:

[0256] Select 6-8 weeks old, SFP grade female C57BL mice and randomly divide them into 4 groups: normal control group (Control group), disease control group (BLM group), TC-2D treatment group (MSC T group), E / N-2D treatment group (MSC EN The mice in the normal control group were injected with 100 μL of normal saline on the back every day, and the mice in the disease control group and cell therapy group were injected with 100 μL of 1 mg / mL bleomycin every day for 28 consecutive days.

[0257] The cells were injected into the tail vein at a dose of 100,000 cells / mouse. Treatment began after the model was successfully established, with injections once a week for a total of 2 times. Samples were collected 28 days after the model was established to detect the treatment effect.

[0258] 1.2 Cell co-culture model

[0259] Co-culture with mouse fibroblasts: Mouse fibroblasts were inoculated in the lower chamber of the transwell. When the cells grew to 70-80% confluence, 10 ng / mL TGF-β was used to construct a fibrosis group, and the cells were inoculated in the upper chamber. The PCR and WB methods were used to study its fibrosis reversal function. The PCR method was based on the method of Example 2, and the following primers were used to perform PCR to detect the expression of related genes:

[0260]

[0261] Co-culture with PBMC: The bottom of the cell culture plate was treated with low adhesion, and the cells and PBMC were inoculated in a 48-well plate at a ratio of 1:10. The PBMC proliferation inhibition level was detected after 3 days.

[0262] 2. Results

[0263] Under three-dimensional conditions, MSC EN Cells can inhibit the proliferation of activated T cells, MSC EN The inhibition efficiency of cells was increased by about 30% compared with the Control group. Fig.37 MSC EN Reversed the fibrotic phenotype of skin fibroblasts and inhibited the expression of fibrosis-related genes, Col1A1 downregulated by about 25% and α-SMA downregulated by about 17%. Fig.38 . With MSC T Compared with the MSC group EN The cells slowed down the immune infiltration in the lesion site (pro-inflammatory CD4+ lymphocytes decreased by 6.5%, Th1 lymphocytes were suppressed by 0.17%, and anti-inflammatory regulatory T lymphocytes Treg cells were enhanced by 0.2%). Fig.39 MSCEN The cells reversed the degree of fibrosis of skin fibroblasts (the thickness of the dermis decreased by about 20%, and the thickness of the subcutaneous fat layer increased by about 1.5 times), such as Fig.40 MSC EN The cells effectively reduced collagen deposition in lung tissue and reversed the degree of tissue fibrosis (down-regulating COL1A1 and COL1A2 by about 30%, down-regulating COL3A1 by about 25%, and down-regulating α-SMA by about 20%). Fig.41 MSC EN The cells significantly improved the integrity and permeability of blood vessels, such as Fig.42 .

[0264] Embodiment 7, MSC EN Treating Sjogren's disease

[0265] 1. Modeling and treatment of Sjögren's disease mice:

[0266] ① Select 8-week-old, SFP-grade female NOD mice, feed them under constant temperature conditions without special pathogens, and examine whether they are diseased by measuring the saliva flow rate.

[0267] ②After the disease is confirmed, MSCs T and MSC EN All mice were treated once with a dose of 100,000 cells / mouse via tail vein injection, and samples were collected 21 days after model establishment.

[0268] 2. Results

[0269] MSC EN The cells improve the salivary gland function of Sjögren's disease, increase the salivary gland flow rate, improve the salivary gland / body weight ratio, reduce salivary gland damage and collagen deposition, reduce inflammatory cell infiltration, reduce acinar and glandular duct atrophy, reduce fibrosis, increase the expression of aquaporin, and relieve salivary gland function. Fig.43 .

[0270] In summary, the present invention provides an application of matrix-enabled mesenchymal stem cells in the preparation of drugs for the prevention and treatment of immune diseases. The mesenchymal stem cells are obtained by culturing and enabling the cell culture matrix hENFc, and the cell culture matrix hENFc is prepared from 1-3 parts of hN-cad-Fc and 1-3 parts of hN-cad-Fc. The matrix-enabled mesenchymal stem cells can promote the nuclear transfer of β-catenin, enhance the proliferation capacity of in vitro expansion, the ability to maintain stemness, and delay cell aging and apoptosis. The hMSCs cultured to the eleventh generation through hENFc empowerment are not tumorigenic and have no adverse effects on animals, meeting the clinical application standards of hMSCs. The matrix-enabled mesenchymal stem cells not only have better survival ability in the lesion microenvironment, but also have stronger environmental perception and anti-inflammatory ability, and significantly inhibit PBMC proliferation ( Figure 5 A) and promote cell apoptosis ( Figure 5 B), MSC EN It also inhibits the proportion of CD4+ and CD8+ T cell subsets, and improves the efficacy of hMSCs in inflammatory and autoimmune diseases. The matrix-empowered mesenchymal stem cells can treat systemic lupus erythematosus nephritis. After treatment, the thickening of the glomerular mesangium and basement membrane, inflammatory cell infiltration, interstitial fibrosis and occasional crescent formation in mice were significantly reduced; the matrix-empowered mesenchymal stem cells can treat diabetic nephropathy. After treatment, the pathological symptoms of the renal tissues of mice were alleviated, and the progression of diabetic nephropathy was significantly delayed; the increase in blood glucose, 24-hour urine protein, BUN and SCr in rats was significantly inhibited; the potential of hMSCs to slow down kidney damage in diabetic rats was effectively improved, the renal function of DKD rats was improved, the cell damage caused by oxidative stress was effectively reduced, the development of diabetic nephropathy was alleviated, and it has a stronger anti-apoptosis ability. The matrix-empowered mesenchymal stem cells can inhibit the proliferation of activated T cells, MSC EN Cells compared to MSC T The inhibition efficiency of the group was increased by about 30%, the fibrotic phenotype of skin fibroblasts was reversed, the expression of fibrosis-related genes was inhibited, Col1A1 was downregulated by about 25%, α-SMA was downregulated by about 17%, the immune infiltration of the lesion site was slowed down (pro-inflammatory CD4+ lymphocytes decreased by 6.5%, Th1 lymphocytes were inhibited by 0.17%, and anti-inflammatory regulatory T lymphocytes Treg cells were enhanced by 0.2%), and the degree of fibrosis of skin fibroblasts was reversed (the thickness of the dermis was reduced by about 20%, and the thickness of the subcutaneous fat layer was increased by about 1.5 times); the collagen deposition of skin and lung tissues was effectively reduced, and the degree of tissue fibrosis was reversed (COL1A1 and COL1A2 were downregulated by about 30%, COL3A1 was downregulated by about 25%, and α-SMA was downregulated by about 20%); and the integrity and permeability of blood vessels were significantly improved. The matrix-empowered mesenchymal stem cells can improve the salivary gland function of Sjogren's disease, increase the salivary gland flow rate, reduce inflammatory cell infiltration, reduce atrophy of alveoli and glandular ducts, reduce the degree of fibrosis, increase the expression of aquaporin, and relieve the salivary gland function.

Claims

1. Use of matrix-empowered mesenchymal stem cells in the preparation of drugs for preventing and treating immune and metabolic diseases and related tissue damage, characterized in that: The matrix-enabled mesenchymal stem cells are obtained by culturing and enabling mesenchymal stem cells in a cell culture matrix hENFc, and the cell culture matrix hENFc is prepared from 1-3 parts of hN-cad-Fc and 1-3 parts of hN-cad-Fc.

2. The use according to claim 1, characterized in that The cell culture matrix hENFc is diluted with hE-cad-Fc and hN-cad-Fc to a total protein concentration of 1-30 μg / mL, added to a tissue culture treated plate, incubated for 30-150 minutes, and washed.

3. The use according to claim 1, characterized in that The immune diseases include autoimmune diseases and inflammatory immune diseases.

4. The use according to claim 3, characterized in that The autoimmune diseases include one or more of systemic lupus erythematosus, systemic sclerosis, Sjögren's disease, hyperthyroidism, juvenile diabetes, diabetic nephropathy, idiopathic thrombocytopenic purpura, and autoimmune hemolytic anemia; the inflammatory immune diseases include one or more of lupus nephritis, rheumatoid arthritis, systemic vasculitis, ulcerative colitis, chronic hepatitis, and polyneuritis.

5. The use according to claim 1, characterized in that The mesenchymal stem cells are derived from mammals.

6. The use according to claim 5, characterized in that The mesenchymal stem cells are derived from humans, pigs or mice.

7. The use according to claim 6, characterized in that The mesenchymal stem cells are derived from bone marrow, adipose tissue, dental pulp, umbilical cord, umbilical cord blood, amniotic fluid or placenta.

8. The use according to claim 7, characterized in that The mesenchymal stem cells are derived from umbilical cord or umbilical cord blood.

Images