Matrix and cytokine combined energized mesenchymal stem cell aggregate and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a mesenchymal stem cell aggregate jointly energized by matrix and cell factors and application of the mesenchymal stem cell aggregate. The aggregate is prepared by culturing mesenchymal stem cells in a culture medium added with a cell culture matrix and IGF-1 (Insulin-like Growth Factor-1) to obtain pretreated mesenchymal stem cells; and continuing subculture in a 3D cell culture system to obtain the cadherin cell culture medium, wherein the cadherin cell culture medium is prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc. The obtained aggregate is complete in structure and clear in boundary, cell endogenous cadherin and phosphorylated IGF-1 receptors are highly expressed, and the curative effect of treating refractory and repeated immune disorder diseases, such as systemic sclerosis, xerosis and systemic lupus erythematosus, with complex etiology is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a mesenchymal stem cell aggregate jointly empowered by a matrix and cytokines and an application thereof. Background Art

[0002] Stem cell therapy is the most promising disease treatment method in the 21st century. It regenerates damaged cells and tissues in the human body through the self-renewal, differentiation and paracrine properties of stem cells. Among them, mesenchymal stem cell (MSC) therapy accounts for 46% of the total number of stem cell clinical trials, covering a variety of indications including degenerative diseases, autoimmune diseases, organ fibrosis and cancer, and has become a hot spot for translational research in the next generation of cell therapy. However, the therapeutic function of MSC in most clinical stages is not as effective as demonstrated in preclinical studies, and the expected therapeutic effect cannot be achieved. In addition to the heterogeneity of MSC and the functional decline caused by its limited in vitro expansion ability, another important reason is the cell survival and functional response caused by environmental changes after in vivo entry. The problem of MSC heterogeneity and functional decline caused by in vitro expansion has been basically solved by the cell empowerment culture system constructed in our laboratory in the early stage. However, how to improve its environmental responsiveness, survival and therapeutic potential after entering the body through pre-induction in the in vitro expansion stage is an urgent problem to be solved in the clinical application of MSC.

[0003] In order to solve this problem, a variety of empowerment culture technologies have been developed to improve the tolerance, responsiveness and therapeutic potential of MSCs entering the lesion microenvironment. Common MSC empowerment strategies include hypoxia treatment, 3D culture, and the addition of nutritional factors (growth factors, cytokines or hormones), which promote the in vitro expansion of MSCs and improve the therapeutic function of MSCs. In recent years, with the development of tissue engineering, tissue engineering scaffold materials (including hydrogels, porous scaffolds, microcarriers, etc.) have been used to load MSCs and implant them into the lesion site, showing the function of engineered MSCs in promoting the repair of damaged tissues. Our research group has also developed a series of calcium-adherin-modified biomaterial microspheres to mediate MSC aggregation. The "one-pot method" prepared composite aggregates of material microspheres and MSCs. The results showed that the differentiation and secretion characteristics of MSCs can be effectively regulated, and it has good application prospects in the field of organoid construction and local tissue damage repair such as cartilage, blood vessels, and colorectal cancer. However, the precise regulation of the biocompatibility and biodegradability of exogenous tissue engineering scaffold materials cannot fully meet the requirements of stem cell therapy technology and stem cell drug development.

[0004] On the basis of previous studies, the research group to which the inventor belongs found through experimental studies that compared with commonly used MSC-activating cytokines (such as IL1-β, IFN-γ or TNF-α), the artificial extracellular matrix of the calcium-adherin fusion protein combined with E-Cad-Fc and N-cad-Fc can more efficiently activate the biological activities such as proliferation and secretion of MSC in collaboration with IGF-1. They innovatively developed a technology for empowering the expansion of MSCs with the artificial extracellular matrix of the calcium-adherin fusion protein combined with IGF-1, and further used 3D culture technology to creatively realize the "two-step method" for preparing pure cell aggregates of empowered MSCs (i.e., empowered MSC aggregates without scaffold materials), which not only reduces the anoikis of MSCs, but also significantly improves the expression of the therapeutic functions of MSCs for immune and inflammatory diseases such as lesion microenvironment tolerance, immune regulation and anti-inflammatory, providing a revolutionary new technology for the regulation of cell function for the treatment of autoimmune and inflammatory diseases and the development of cellular drugs. Summary of the invention

[0005] The primary purpose of the present invention is to provide a mesenchymal stem cell aggregate jointly empowered by matrix and cytokines, wherein the aggregate is obtained by culturing mesenchymal stem cells in a culture medium supplemented with cell culture matrix and IGF-1 to obtain pretreated mesenchymal stem cells, and continuing to culture in a 3D cell culture system, wherein the cell culture matrix is ​​prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc.

[0006] Preferably, the matrix and cytokine-combined mesenchymal stem cell aggregates are prepared by the following method: (1) diluting 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc to a total protein concentration of 1-30 μg / mL, adding to a tissue culture treated cell culture plate, incubating, and washing to obtain a cell culture matrix;

[0007] (2) inoculating the mesenchymal stem cells into the cell culture matrix-enhanced cell culture plate obtained in step (1), and continuously subculturing to obtain matrix-empowered mesenchymal stem cells;

[0008] (3) culturing the matrix-empowered mesenchymal stem cells obtained in step (2) until the cells grow to 50-60% confluence, adding 0-15 ng / mL of human recombinant IGF-1, culturing until 70-80% confluence, digesting, and collecting the cells;

[0009] (4) The cells obtained in step (3) are added to a 3D cell culture system and cultured to obtain mesenchymal stem cell aggregates jointly empowered by matrix and cytokines.

[0010] Preferably, the incubation time in step (1) is 0.5-24 hrs, and the incubation temperature is 4-37°C.

[0011] Preferably, the culture medium used in step (3) is a complete culture medium for MSC in vitro expansion, with additional addition of IGF-1.

[0012] The second object of the present invention is to provide a method for preparing the mesenchymal stem cell aggregates pretreated with cadherin combined with IGF-1, wherein the matrix and cytokine-combined mesenchymal stem cell aggregates are prepared by the following method: (1) diluting 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc to a total protein concentration of 1-30 μg / mL, adding them to a tissue culture treated cell culture plate, incubating, and washing to obtain a cell culture matrix;

[0013] (2) inoculating the mesenchymal stem cells into the cell culture matrix-enhanced cell culture plate obtained in step (1), and subculturing them to obtain matrix-empowered mesenchymal stem cells;

[0014] (3) culturing the matrix-empowered mesenchymal stem cells obtained in step (2) until the cells grow to 50-60% confluence, adding 0-15 ng / mL of human recombinant IGF-1, culturing until 70-80% confluence, digesting, and collecting the cells;

[0015] (4) The cells obtained in step (3) are added to a 3D cell culture system and cultured to obtain mesenchymal stem cell aggregates jointly empowered by matrix and cytokines.

[0016] The third object of the present invention is to provide the use of the matrix and cytokine-combined mesenchymal stem cell aggregates in the preparation of drugs for treating immune diseases.

[0017] Preferably, the immune diseases include autoimmune diseases and inflammatory immune diseases.

[0018] 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.

[0019] The beneficial effects of the present invention are as follows: (1) The present invention provides a matrix and cytokine-combined mesenchymal stem cell aggregate (E / N-IGF-3D), wherein the matrix and cytokine-combined mesenchymal stem cell aggregate is obtained by culturing pretreated mesenchymal stem cells in a 3D cell culture system, wherein the pretreated mesenchymal stem cells are obtained by culturing mesenchymal stem cells on a cadherin cell culture matrix and in a culture medium supplemented with IGF-1, wherein the cadherin cell culture matrix is ​​prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc, and the aggregate cell structure obtained by combined pretreatment with E / N-cad and 0-15ng / mL IGF-1 is complete and has clear boundaries. The expression of endogenous cadherin, IGF-1 receptor and its phosphorylated receptor of E / N-IGF-3D cells is significantly increased, the anti-inflammatory function is significantly upregulated, and the cell metabolic pathway is more biomimetic to the metabolic form of MSC in vivo, which is conducive to improving the expression of therapeutic function after cell reinfusion. In vitro cell experiments showed that E / N-IGF-3D aggregates can effectively inhibit the proliferation of activated T cells, efficiently reverse the fibrotic phenotype of skin fibroblasts, promote the repair of damaged endothelial cells, and improve the integrity of the vascular endothelium.

[0020] (2) When the E / N-IGF-3D aggregates described in the present invention are used to treat systemic sclerosis, the E / N-IGF-3D aggregate treatment group effectively inhibits the immune infiltration of lymphocytes and monocytes in the lesion site, reduces pro-inflammatory CD4+ lymphocytes, and increases anti-inflammatory Treg cells; at the same time, the E / N-IGF-3D aggregates have a significant therapeutic effect of reversing dermal fibrosis and pulmonary fibrosis; in addition, the E / N-IGF-3D aggregates significantly improve the vascular integrity and permeability of fibrotic lung tissue.

[0021] (3) When the E / N-IGF-3D aggregates of the present invention are used to treat Sjögren's disease, the E / N-IGF-3D aggregate treatment group significantly up-regulated salivary gland flow rate, increased salivary gland / body weight ratio, reduced inflammatory cell infiltration, reduced acinar and duct atrophy, reduced salivary gland collagen deposition and fibrosis, and increased the expression of the water channel protein AQP5 and the ion transporter protein NKCC1 in damaged salivary glands, effectively improving the salivary gland function of Sjögren's disease;

[0022] (4) When the E / N-IGF-3D aggregates described in the present invention are used to treat systemic lupus erythematosus in MRL / Lpr mice, the E / N-IGF-3D aggregate treatment group more effectively inhibits enlargement of perinephric lymph nodes and spleen, and inhibits lymphocyte proliferation; significantly reduces the physiological and biochemical indicators of systemic lupus erythematosus mice, including the content of protein, creatinine, and urea nitrogen in urine, and various pathological parameters such as anti-dsDNA antibody levels, TNF-α and IgG concentrations in blood; and the pathological characteristics of systemic lupus erythematosus mice in the E / N-IGF-3D aggregate treatment group, such as thickening of the glomerular mesangium and basement membrane, inflammatory cell infiltration, interstitial fibrosis, and occasional crescent formation, are significantly reduced; at the same time, E / N-IGF-3D aggregates more effectively inhibit inflammation by regulating immune cells, further improving the renal damage of MRL / Lpr mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 Preparation of hENFc 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 Figure 2 Characterization of hENFc Note: (A) Element analysis on different substrate surfaces; (B) Stability test on different substrates; (C) Distribution and structure test of hE-cad-Fc and hN-cad-Fc on different substrate surfaces; (D) Hydrophilicity test on different substrate surfaces

[0025] Figure 3 Detection of the proliferation ability of hMSCs at different passages on different matrix surfaces

[0026] Note: (A) cell doubling time; (B) cell number

[0027] Figure 4 Statistics of hMSCs volume at different generations on different matrix surfaces

[0028] Figure 5 Detection of endogenous cadherin expression in hMSCs of different passages on different matrix surfaces

[0029] Note: (A) Fluorescence quantitative PCR detection; (B) Western Blotting detection

[0030] Figure 6Expression of OCT4 gene and protein in hMSCs of different passages on different matrices

[0031] Figure 7 Senescence detection of hMSCs at different passages on different matrix surfaces

[0032] Note: (A) Cell cycle detection; (B) β-galactosidase staining

[0033] Figure 8 Apoptosis detection of hMSCs at different passages on different matrices

[0034] Fig. 9 Multiple differentiation ability tests of hMSCs of different generations on different matrix surfaces Note: (A) Oil red O staining and adipogenic gene expression test; (B) Alizarin red staining and osteogenic gene expression test; (C) Alcian blue staining and chondrogenic gene expression test

[0035] Fig.10 MSC EN Oncogene and tumor suppressor gene expression dot plots

[0036] Fig.11 MSC EN Karyotype analysis

[0037] Fig.12 MSC EN Tumorigenicity assay

[0038] Note: (A) Light microscopic image of mouse tumor size; (B) Mouse weight; (C) Tumor volume

[0039] Fig.13 Effects of E / N-cad combined with different factors pretreatment on MSC function

[0040] Fig.14 Effects of different concentrations of IGF pretreatment on cell proliferation

[0041] Fig.15 Effects of different concentrations of IGF pretreatment on endogenous cadherin, stemness and anti-inflammatory function of cells

[0042] Fig.16 Expression levels of cadherin, IGF-1 receptor, anti-inflammatory, chemotactic and regeneration-related genes after combined treatment of E / N-cad and IGF-1

[0043] Fig.17 E / N-IGF-3D aggregate morphology

[0044] Fig.18 Expression levels of endogenous cadherin, IGF-1 receptor and stemness genes in E / N-IGF-2D and E / N-IGF-3D aggregates

[0045] Fig.19 Expression and phosphorylation activation level of IGF-1 receptor in E / N-IGF-3D aggregates

[0046] Fig. 20 Expression levels of genes related to anti-inflammatory, glucose metabolism and mitochondrial function in E / N-IGF-3D aggregates

[0047] Fig.21 Morphology and size distribution of exosomes secreted by E / N-IGF-3D aggregates

[0048] Fig. 22 Expression levels of marker proteins in exosomes secreted by E / N-IGF-3D aggregates

[0049] Fig. 22 Expression levels of anti-inflammatory and anti-fibrotic miRNAs in E / N-IGF-3D aggregates

[0050] Fig.24 Survival ability of E / N-IGF-3D aggregates under inflammatory conditions (A) Live / dead staining; (B) CCK8 detection

[0051] Fig.25 Anti-apoptotic ability of E / N-IGF-3D aggregates under inflammatory conditions

[0052] Fig.26 E / N-IGF-3D aggregates inhibit the proliferation of activated PBMCs

[0053] Fig. 27 E / N-IGF-3D aggregates promote the repair of damaged endothelium (A) Flow cytometry of damaged endothelial cell apoptosis; (B) Statistics of damaged endothelial cell apoptosis; (C) WB detection of damaged endothelial cell phenotype and apoptosis markers; (D) PCR detection of damaged endothelial cell phenotype

[0054] Fig.28 E / N-IGF-3D aggregates inhibit fibrosis

[0055] Fig.29 E / N-IGF-3D aggregates inhibit inflammatory infiltration in systemic sclerosis lesions

[0056] Fig.30 E / N-IGF-3D aggregates inhibit fibrosis in systemic sclerosis lesional skin

[0057] Fig.31 E / N-IGF-3D aggregates inhibit pulmonary fibrosis in systemic sclerosis lesions

[0058] Fig.32 E / N-IGF-3D aggregates improve vascular integrity in systemic sclerosis lesions

[0059] Fig.33 E / N-IGF-3D aggregates improve vascular permeability in systemic sclerosis lesions

[0060] Fig.34 Effect of E / N-IGF-3D aggregates in the treatment of Sjögren's disease (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)

[0061] Fig.35 Morphological study of E / N-IGF-3D aggregates in the treatment of systemic lupus erythematosus mice

[0062] Note: (A) Hair observation; (B) Spleen morphology and index; (C) Perinephric lymph node morphology and index

[0063] Fig.36 Physiological and biochemical indicators of systemic lupus erythematosus mice treated with E / N-IGF-3D aggregates Note: (A) Urine protein level; (B) Creatinine level; (C) Urea nitrogen level; (D) Blood anti-dsDNA antibody level; (E) Blood TNF-α content; (F) Blood IgG level

[0064] Fig.37 Pathological staining and scoring of kidneys in mice with systemic lupus erythematosus treated with E / N-IGF-3D aggregates

[0065] Note: (A) Renal pathology staining; (B) Renal score; (C) Immune complex immunofluorescence staining

[0066] Fig.38 Immune cell analysis of SLE mice treated with E / N-IGF-3D aggregates Note: (A) Splenic Treg cell ratio; (B) Perirenal lymph node Treg cell ratio; (C) Splenic CD4 + T cell ratio; (D) CD4 + T cell ratio; (E) CD11b+ cell effector T cell ratio DETAILED DESCRIPTION

[0067] 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.

[0068] In the following examples, unless otherwise specified, all reagents used can be purchased from the market.

[0069] In the following examples, unless otherwise specified, the methods used are all conventional methods.

[0070] 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.

[0071] The matrix jointly modified by hE-cad-Fc and hN-cad-Fc is referred to as hENFc.

[0072] 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.

[0073] Example 1. Preparation of hENFc matrix and novel mesenchymal stem cells

[0074] 1. Preparation of hENFc Matrix

[0075] Purified hE-cad-Fc and / or hN-cad-Fc were diluted in PBS to a total protein concentration range of 1-30 μg / mL, and 150 μL of protein solution per square centimeter was added to tissue culture treated plates (TCPS); after incubation at 4-37°C for 0.5-24 hrs, the matrix was further washed 5 times with PBS.

[0076] 2. Detection of adsorption amount-concentration and adsorption amount-time of hE-cad-Fc and hN-cad-Fc

[0077] 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 120 min, the plate was further washed 5 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 5 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 with PBS five times, 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.

[0078] 3. Hydrophilicity detection, elemental analysis and morphology detection of hENFc matrix

[0079] 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).

[0080] 4. Stability test of hENFc matrix

[0081] Purified hE-cad-Fc and hN-cad-Fc were diluted in PBS to a total concentration of 1-30 μg / mL, and 100 μL per well was added to a 96-well plate. After incubation at 4-37°C for 0.5-24 hrs, the plate was further washed 5 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.

[0082] 6. Results

[0083] 6.1 Identification of hN-cad-Fc, hE-cad--Fc and preparation of hENFc

[0084] 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).

[0085] 6.2 Physicochemical Characterization of hENFc Matrix

[0086] 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.

[0087] 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.

[0088] Example 2: Preparation and function of hENFc matrix-enabled hMSCs

[0089] 1. Preparation of hENFc matrix-enhanced hMSCs

[0090] 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.

[0091] 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 .

[0092] 2. Detection of the proliferation ability of hENFc matrix-enabled hMSCs

[0093] All cells on the surface of different matrices collected 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 determine cell viability after incubation for 24 and 48 hours. The CCK8 viability detection method is as follows: discard the supernatant and wash the cells 5 times with PBS. Add 100 μL of CCK-8 (1:10 dilution) reagent to each well, incubate at 37°C for 4 hours, and detect the absorbance at 450nm using an enzyme reader. 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:

[0094]

[0095]

[0096] 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.

[0097] 3. Detection of stemness of hMSCs empowered by hENFc matrix

[0098] MSCs were harvested at passages 6, 11, and 15. T and MSC EN Total RNA was extracted by the following method: all culture medium was discarded, cells were washed 3 times with PBS, 1 mL of Trizol solution was added to each well to lyse the cells, and then the cells were transferred to a 1.5 mL centrifuge tube, 200 μL of chloroform was added to shake vigorously for 15 sec, incubated at room temperature for 5 min, and centrifuged at 4°C and 12000 g for 15 min; the supernatant was aspirated into a new centrifuge tube, 500 μL of isopropanol was added, incubated at room temperature for 10 min, and centrifuged at 4°C and 12000 g for 10 min; after discarding the supernatant, 1 mL of 75% ethanol was added to each tube, mixed well, and centrifuged at 4°C and 7500 g for 5 min, the supernatant was discarded, and the tubes were dried at room temperature; finally, 20 μL of sterile and enzyme-free ddH2O was added to each tube to dissolve the RNA, the tubes were water bathed at 55°C for 10 min, and the tubes were aliquoted and stored in a -80°C ultra-low temperature refrigerator. The total protein was extracted by the following method: all culture medium was discarded, and then the cells were washed with PBS. 100 μL of strong lysis buffer was added to each well and incubated on ice for 10 min. All liquid was scraped off by cell scraping and transferred to a centrifuge tube. The tube was centrifuged at 4°C and 13,000 g for 15 min, and the supernatant was taken. The protein concentration in each sample was determined according to the instructions of the BCA kit. Finally, loading buffer (1:5 dilution) was added to the supernatant of each tube and boiled for 10 min. After aliquoting, the tube was stored in a -80°C ultra-low temperature refrigerator.

[0099] The expression of E-cadherin, N-cadherin and OCT4 genes and proteins was detected by fluorescence quantitative PCR and Western Blotting. The fluorescence quantitative PCR method is as follows: according to the Vazyme instructions, 2 μL of cDNA of each group was added to each tube as a template, and 1 μL of primers were added respectively. In addition, each group was supplemented with 10 μL 5×ChamQ Universal SYBR qPCR Master Mix and 7 μL RNase-free water; after the addition of the sample, it was centrifuged for 5 seconds using a handheld centrifuge, placed in a fluorescence quantitative PCR instrument and amplified according to the program of 95℃ 30sec, 95℃ 3sec, 60℃ 10sec repeated 40 times, 95℃ 15sec, 60℃ 60sec, 95℃ 15sec; the data obtained used the expression of β-actin in each group as the internal reference, and three groups were set up in parallel for each sample, using 2 -ΔΔCt The Western Blotting method is as follows: add the sample to 10% SDS-polyacrylamide gel for electrophoresis analysis. When the sample front migrates to the distance from the bottom of the glass plate, the electrophoresis is terminated; take out the PAGE gel, use the transfer clip to clamp the absorbent sponge, PAGE gel, PVDF membrane, and absorbent sponge in order, place them in the electrotransfer instrument, add the electrotransfer buffer and electrotransfer at 100V for 90 minutes; after the transfer is completed, take out the PVDF membrane and block it with 5% BSA at room temperature for 2 hours; then incubate with the primary antibody at 37°C for 2 hours or 4°C overnight, then wash with TBST solution at room temperature for 5 minutes, repeat three times; add HRP-labeled secondary antibody and incubate at room temperature for 2 hours; then wash with TBST solution at room temperature for 5 minutes, repeat 5 times; finally, add an appropriate amount of color developing solution on the PVDF membrane, observe and take pictures with the Western Blotting exposure instrument, and use Image J for statistical analysis of the graphics.

[0100] The primer sequences used in the implementation are as follows:

[0101]

[0102] 4. Cycle detection of hENFc matrix-enhanced hMSCs

[0103] At the 6th, 11th and 15th passages, 1×10 6 MSC T and MSC ENThe cells were fixed in 70% cold ethanol for 2 hrs 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, and the cells were resuspended and 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.

[0104] 5. Senescence detection of hENFc matrix-enhanced hMSCs

[0105] 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 five 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.

[0106] 6. Apoptosis detection of hENFc matrix-enhanced hMSCs

[0107] 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, unstained cells are used as blank tubes; the tubes without propidium iodide and only AnnexinV / FITC are used as single-stained tubes.

[0108] 7. Detection of differentiation potential of hMSCs empowered by hENFc matrix

[0109] 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 DMEM / F12 supplemented with 50 μg / mL ascorbic acid 2-phosphate, 10 nM dexamethasone (Dex), 10 mM β-glycerophosphate, 10% FBS, and 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 DMEM / F12 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 DMEM / F12 supplemented with 2% FBS, 50 μg / mL ascorbic acid 2-phosphate, 50 μg / mL L-proline, 100 nm Dex, 10ng / mL TGF-β1, 1% human insulin, transferrin and sodium selenite were added to culture cells in DMEM / F12 to induce chondrogenesis, and toluidine blue chondrogenic solution was used to detect mature cells. At the same time, referring to the method of Example 2, RNA was extracted and converted into cDNA, and PCR was performed using the following primers to detect the expression of genes related to the three-phase differentiation:

[0110]

[0111] 8. Detection of tumorigenicity of hENFc matrix-enhanced hMSCs

[0112] To examine the tumorigenicity of hENFc-enabled hMSCs, 1×10 7 6th generation MSC T (n=3) and 15th 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.

[0113]

[0114] 9. Results

[0115] 9.1 Effect of hENFc matrix on the proliferation of hMSCs expanded in vitro

[0116] 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 3 However, hMSCs (MSCT ), 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 25h 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 ).

[0117] 9.2 Effect of hENFc matrix on the maintenance of stemness of hMSCs expanded in vitro

[0118] 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. E-cadherin plays a key role in maintaining the stemness of stem cells. We continued to investigate the expression of hMSCs stemness indicator OCT4 in long-term passage. Gene and protein detection results showed that, compared with MSC 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 6 )

[0119] 9.3 Effects of hENFc matrix on senescence and apoptosis of hMSCs expanded in vitro

[0120] 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 theT ( Figure 7 ). The result is Figure 8 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.

[0121] 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. 9 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. 9 B) and cartilage ( Fig. 9 C) differentiation potential.

[0122] 9.3 Safety Analysis of hENFc Matrix-Enabled hMSCs

[0123] 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.10), 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.11 ).

[0124] 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 MSC cultured to the fifteenth 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.12 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.12 B) Seeding MSCs T and MSC EN No bulge formed at the inoculation site of the mice. Fig.12 C) These results indicate that hMSCs cultured to the fifteenth generation after hENFc empowerment are not tumorigenic and have no adverse effects on animals, which meets the clinical application standards of hMSCs.

[0125] Example 3: Preparation of cell aggregates pretreated with cadherin combined with IGF-1

[0126] 1. Methods

[0127] 1.1 Preparation of cells pretreated with cadherin combined with IGF-1:

[0128] E-cadherin matrix-enabled MSCs were cultured in complete medium. After 48-72 hrs, the cells grew to 50-60% confluence. The culture medium was pretreated with human recombinant IGF-1 at a concentration of 0-15 ng / mL for 10-24 hrs. The pretreated cells were collected when the cells grew to 70-80% confluence. Complete medium without human recombinant IGF-1 and complete medium supplemented with IL-1β, IFN-γ and TNF-α (at a concentration of 10 ng / mL, the concentration commonly used for MSC activation in the literature) were used as the control group.

[0129] 1.2 Preparation of cell aggregates pretreated with cadherin combined with IGF-1:

[0130] E-cadherin matrix-enabled MSCs were cultured in complete medium, and the cells grew to 50-60% confluence in 48-72 hrs. The cells were pretreated with 0-15 ng / mL human recombinant IGF-1 medium for 10-24 hrs. The cells grew to 70-80% confluence, digested and collected, and 360,000 cells / well (300 cells / aggregate) of E / N-cad+IGF-1-3D cell aggregates (referred to as E / N-IGF-3D aggregates) were prepared in AggreWell cell culture plates.

[0131] 1.3 Western blotting

[0132] Proteins were extracted from cells and cells in three-dimensional culture were extracted using an ultrasonic disruptor. Proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. Nonspecific binding sites on the PVDF membrane were blocked with skim milk, followed by incubation of the primary antibody with the target protein, followed by washing to remove unbound antibodies. Secondary antibodies were then incubated to bind to the antibodies immunoreactive with the primary antibody, and unbound secondary antibodies were washed again to remove unbound secondary antibodies. Fluorescent substrates were added for chemiluminescent reaction to observe the signal of the target protein, and the protein signal was captured using a Western blot imaging system, and the signal was quantitatively analyzed using image analysis software to compare the protein expression levels under different conditions.

[0133] 1.4 Fluorescence quantitative PCR

[0134] Referring to the method of Example 2, RNA was extracted and converted into cDNA, and PCR was performed using the following primers to detect the expression of genes related to anti-inflammation, tissue regeneration, stemness, metabolism, and exosome secretion:

[0135]

[0136] 2. Results

[0137] 2.1 Optimization of IGF-1 pretreatment conditions

[0138] Using different factors combined with E / N-cad to pretreat MSCs, the results showed that IGF-1 can significantly improve the secretory function of MSCs compared with other factors, such as Fig.13 .

[0139] When the cells were cultured to 50-60% confluence, different concentrations of human recombinant IGF-1 were added to the culture medium. It was shown that when the concentration of IGF-1 increased to 10 ng / mL, the cell proliferation effect was significant and stable, such as Fig.14 .

[0140] IGF-1 pretreatment can regulate the expression of anti-inflammatory and stemness genes in cells. The results of the concentration optimization of IGF-1 combined with E / N-cad further showed that compared with the system without adding IGF-1, when the IGF-1 concentration was 10 ng / mL, the Nanog gene level of the cells was upregulated by about 2.5 times, and the anti-inflammatory gene IDO was upregulated by about 70%. Fig.15 Moreover, compared with the TCPS and E / N-cad groups without IGF-1, the E / N-cad combined with 10 ng / ml IGF-1 group showed a significant upregulation of the mRNA expressions of endogenous cadherin, IGF-1 receptor, anti-inflammatory, chemotaxis, repair and regeneration, and homing-related mRNAs, such as Fig.16 .

[0141] Cells pretreated with E / N-cad and 10 ng / mL IGF-1 were prepared into cell aggregates, which had complete structures and clear boundaries. Fig.17 .

[0142] After the cells were pretreated with E / N-cad and IGF-1 and prepared into aggregates, the endogenous E-cadheirn and N-cadheirn, as well as IGFR expression of E / N-IGF-3D cells were higher than that of the aggregates of cells cultured in 2D and TCPS. At the same time, the expression level of the cell stemness gene Nanog was significantly increased. Fig.18 .

[0143] Compared with TC-3D, TC-IGF-3D and E / N-3D, the expression of IGF-1 receptor and its phosphorylated receptor in E / N-IGF-3D cell aggregates was significantly increased. In particular, compared with TC-IGF-3D, the formation of 3D cell aggregates after E / N-cad combined with IGF-1 pretreatment significantly upregulated the cell's response activity to IGF-1 stimulation, achieving a nonlinear level of upregulation of IGF-1 stimulation responsiveness, such as Fig.19 .

[0144] Compared with the TC-3D group, the expression of the anti-inflammatory gene TSG-6 in the E / N-IGF-3D group was upregulated by about 50%, the gene expression of IL-10 and HGF was significantly upregulated (both more than 4 times), the expression of glycolysis-related genes PFKP and PKM2 was upregulated by about 50%, and the expression of the cell mitochondrial function gene MCU was upregulated by about 50%. Fig. 20 That is, compared with TC-3D, the anti-inflammatory activity of E / N-IGF-3D cell aggregates was significantly upregulated, and its metabolic pathway was more biomimetic to the metabolic form of MSCs in vivo, which was beneficial to improve the expression of therapeutic functions after cell reinfusion.

[0145] Example 4: Exosome extraction and identification

[0146] 1. Methods

[0147] 1.1 Collection of exosomes:

[0148] E / N-IGF-3D MSC aggregates were cultured in basal medium for 48 hours and the supernatant was collected. Exosomes were collected by gradient centrifugation: 300g, 4℃ centrifugation for 10 min to remove cells and take the supernatant; 10,000g, 4℃ centrifugation for 30 min to remove cell debris and take the supernatant; 100,000g, 4℃ centrifugation for 90 min, and the precipitate was collected as exosomes.

[0149] 1.2 Exosome identification and detection:

[0150] ① Dynamic light scattering (DLS) to measure particle size: Take 20 μL of exosome solution resuspended in PBS, dilute it 100 times to 2 mL with PBS prepared with pure water, and place it in a test dish. Then place it in a dynamic light scattering instrument for analysis, and draw a particle size distribution graph with the obtained data results.

[0151] ② Transmission electron microscopy (TEM) exosome morphology determination: Take 10 μL of exosome solution resuspended in PBS and add it to the copper mesh, let it stand at room temperature for 1-2 minutes, use filter paper to absorb the sample liquid along the outer side of the copper mesh, add 10 μL of 20 mg / mL phosphotungstic acid dye solution to the copper mesh, let it stand for 1-2 minutes and then absorb it, observe and take pictures under a transmission electron microscope at a magnification of 100,000.

[0152] ③ Protein marker determination: After BCA determination of protein concentration, 40 μg of total exosome protein was taken for WB detection of characteristic proteins CD9, CD63, and CD81.

[0153] ④ Exosome function detection: Referring to the method of Example 2, extract miRNA and convert it into cDNA, and use the following primers to perform PCR to detect the expression of related genes:

[0154]

[0155] 2. Results

[0156] 2.1 The exosomes produced by E / N-IGF-3D MSC aggregates have uniform particle size and intact membrane structure, with a distribution range of 143-184 nm. Fig.21 .

[0157] 2.2 Compared with the TC-3D group, the exosomes produced by E / N-IGF-3D cell aggregates meet the industry standards for exosomes, with high expression of CD9 / CD63 / CD81, negative Calnexin, and slightly upregulated expression of cadherin in exosomes, such as Fig. 22 .

[0158] 2.3E / N-IGF-3D cell aggregate-derived exosomes significantly upregulated anti-inflammatory and anti-fibrosis-related miRNAs, including mir-21 upregulated by about 60 times, mir-181 upregulated by about 150 times, mir-133b-5p upregulated by about 25 times, mir-19b-3p upregulated by about 100 times, mir-23b-5p upregulated by about 4 times, and mir-223-5p upregulated by about 40 times. Fig.23 .

[0159] Example 5: Systemic Sclerosis

[0160] 1. Experimental Methods

[0161] (1) Modeling and treatment of systemic sclerosis mouse model:

[0162] ① 6-8 week old, SFP grade female mice were selected and randomly divided into 4 groups: normal control group (Control group), disease control group (BLM group), TC-3D treatment group (TC-3D group), E / N-IGF-3D treatment group (E / N-IGF-3D group) (a total of 24 mice, 6 mice in each group). The normal control group mice were injected with 100 μL of normal saline on the back every day, and the disease control group and cell aggregate treatment group were injected with 100 μL of 1 mg / mL bleomycin every day for 28 consecutive days.

[0163] ②TC-3D and E / N-IGF-3D cell aggregates were injected into systemic sclerosis model mice via tail vein at a dose of 100,000 cells / mouse, once a week for 2 consecutive times. Samples were collected 28 days after the first injection and the treatment effect was tested according to clinical specimens.

[0164] (2) In vitro cell co-culture to study the therapeutic function of MSC aggregates

[0165] ① 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 fibrotic cell model, and the cell aggregates were inoculated in the upper chamber. The PCR and WB methods were used to study its function of reversing fibrosis. 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:

[0166]

[0167]

[0168] ② Co-culture with human umbilical vein endothelial cells (HUVEC): HUVEC were inoculated in the lower chamber of the transwell. When the cells grew to 70-80% confluence, 0.1U BLM was used to construct an endothelial cell injury model, and the cell aggregates were inoculated in the upper chamber. The PCR and WB methods were used to study its function of promoting endothelial repair. 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:

[0169]

[0170] ③ Co-culture with PBMC: The bottom of the cell culture plate was treated with low adhesion, and the aggregates and PBMCs were inoculated in a 48-well plate at a ratio of 1:10. After 3 days, the proliferation inhibitory function of the cell aggregates on PBMCs was detected.

[0171] In the above implementation content, referring to the method of Example 2, RNA was extracted and converted into cDNA, and PCR was performed using the following primers to detect the expression of related genes:

[0172] 2. Results

[0173] Compared with the TC-3D, TC-IGF-3D and E / N-3D groups, under inflammatory conditions (30 ng / mL IL-1β), the live-death staining results and CCK8 cell viability assay results showed that the survival rate and cell activity of E / N-IGF-3D cell aggregates were significantly improved, as shown in Figure 2. Fig.24 ; WB results showed that the expression of anti-apoptotic protein BCL-2 in E / N-IGF-3D cell aggregates was significantly upregulated, and the expression of pro-apoptotic protein BAX was significantly downregulated, indicating that the anti-apoptotic ability of E / N-IGF-3D cell aggregates was significantly improved under inflammatory conditions. Fig.25 .

[0174] E / N-IGF-3D cell aggregates can effectively inhibit the proliferation of activated T cells, with an inhibition efficiency of up to about 83%. Fig.26In the co-culture experiment of hMSC and damaged endothelium (pre-treated with 0.1U BLM for 72hrs), the results of flow cytometry, WB and PCR of endothelial cells showed that in the E / N-IGF-3D cell aggregate co-culture group, the apoptosis rate of damaged endothelium was reduced by 23.1%, and the expression levels of endothelial barrier-related endothelial cell adhesion proteins VE-cadherin and ZO-1 were upregulated, and the apoptosis-related BCL-2 (anti-regulation) was significantly upregulated, and BAX (pro-regulation) was significantly downregulated. Fig. 27 , that is, E / N-IGF-3D cell aggregates have the effect of promoting the repair of damaged endothelial cells and improving the integrity of the vascular endothelium. Compared with the TC-3D group, E / N-IGF-3D cell aggregates significantly inhibited the expression of fibrosis-related genes in skin fibroblasts, including COL1A1 down-regulated by about 18.8% and α-SMA down-regulated by about 50%. Fig.28 , showing that E / N-IGF-3D cell aggregates have the function of efficiently reversing the fibrotic phenotype of fibroblasts.

[0175] The model animal treatment experiment showed that compared with the TC-3D group, the E / N-IGF-3D cell aggregate group effectively inhibited the immune infiltration of lymphocytes and monocytes in the lesion site of systemic sclerosis, and the pro-inflammatory CD4+ lymphocytes decreased by 19.8%, of which Th1 lymphocytes decreased by 0.72%, and the anti-inflammatory regulatory T lymphocytes Treg increased by 0.5%. Fig.29 , revealing that E / N-IGF-3D cell aggregates have efficient immune regulation and anti-inflammatory effects. At the same time, the thickness of the dermis in the E / N-IGF-3D cell aggregate group decreased by about 16.7%, and the thickness of the subcutaneous fat layer increased by about 1 times, revealing that E / N-IGF-3D cell aggregates have the effect of reversing dermal fibrosis, such as Fig.30 ; Gene detection of lung tissue fibrosis in the E / N-IGF-3D cell aggregate group showed that the expression of COL1A1, COL1A2 and α-SMA genes was downregulated by about 50%, and the expression of COL3A1 gene was downregulated by about 35%. Tissue sections also showed that the collagen deposition in lung tissue was reduced, such as Fig.31 , revealing that E / N-IGF-3D cell aggregates have a significant therapeutic effect in reversing pulmonary fibrosis. Furthermore, vascular immunofluorescence detection of lung tissue in the E / N-IGF-3D cell aggregate treatment group showed that the vascular integrity of fibrotic lung tissue (such as Fig.32 ) and permeability, (e.g. Fig.33 ) was significantly improved. Overall, it was shown that E / N-IGF-3D cell aggregates have the function and efficacy of reversing the fibrosis process of damaged tissues.

[0176] Example 6: Sjogren's disease

[0177] 1. Experimental Methods

[0178] 1.1 Modeling and treatment of Sjögren's disease mice:

[0179] ① Select SFP-grade female NOD mice, confirm the salivary flow rate at 8 weeks of age, establish the Sjögren's disease mouse model (a total of 24 mice, 6 mice in each group), and raise them in an SFP-grade animal room for use.

[0180] ②TC-3D and E / N-IGF-3D cell aggregates were treated by tail vein injection at a dose of 100,000 cells / mouse. Samples were collected 21 days after the first injection and analyzed according to clinical specimens.

[0181] 2. Results

[0182] Compared with the TC-3D treatment group, the E / N-IGF-3D cell aggregate group significantly improved the salivary gland function of Sjögren's disease, including upregulating the salivary gland flow rate by about 30%, increasing the salivary gland / body weight ratio, reducing inflammatory cell infiltration, reducing acinar and glandular duct atrophy, reducing collagen deposition and fibrosis, and increasing the expression of the water channel protein AQP5 and the ion transporter protein NKCC1 in the damaged salivary glands. The salivary gland function was alleviated. Fig.34 .

[0183] Example 7: Systemic lupus erythematosus

[0184] 1. Experimental Methods

[0185] 1.1E / N-IGF-3D cell aggregates for the treatment of systemic lupus erythematosus model mice (MRL / Lpr mice)

[0186] 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 TC-3D and 1×10 6 The E / N-IGF-3D cell aggregates of 100 μL of cells were collected from the PBS group and the samples were analyzed according to the clinical sample test two weeks after treatment.

[0187] 2. Results

[0188] 2.1 Morphological study of E / N-IGF-3D cell aggregates in the treatment of systemic lupus erythematosus mice

[0189] After 2 weeks of treatment, the hair of the mice in the treatment group gradually returned to normal. The images of the perinephric lymph nodes and spleen and the ratio of their weight to body weight showed that the organ size and organ index of the perinephric lymph nodes and spleen of the mice in the E / N-IGF-3D cell aggregate treatment group were significantly lower than those in the other two groups. Fig.35These results indicate that intravenous implantation of E / N-IGF-3D cell aggregates has an inhibitory effect on the enlargement of perirenal lymph nodes and spleen, which is closely related to the inhibitory function of E / N-IGF-3D cell aggregates in inhibiting lymphocyte proliferation.

[0190] 2.2 Study on physiological and biochemical parameters of systemic lupus erythematosus mice treated with E / N-IGF-3D cell aggregates

[0191] like Fig.36 As shown, various parameters such as protein, creatinine, urea nitrogen content in urine, anti-dsDNA antibody level, TNF-α and IgG concentration in blood of mice treated with E / N-IGF-3D cell aggregates were significantly reduced compared with those of mice treated with PBS and TC-3D. That is, the E / N-IGF-3D group was more effective in improving the physiological and biochemical indicators of mice with systemic lupus erythematosus.

[0192] 2.3E / N-IGF-3D cell aggregates in the treatment of systemic lupus erythematosus mice with renal histopathology and antibody deposition

[0193] like Fig.37 As shown in A and B, the results of renal tissue pathology observation and analysis of MRL / Lpr mice using H&E, PAS and Masson pathological staining showed that compared with the PBS treatment group and the TC-3D treatment group, the glomerular, renal interstitial and perivascular damage of the mice in the E / N-IGF-3D cell aggregate treatment group was significantly reduced, effectively reversing the glomerular damage. It is worth noting that compared with the PBS treatment group and the TC-3D cell aggregate treatment group, the fibrotic features of lupus renal system such as thickening of the glomerular mesangium and basement membrane, inflammatory cell infiltration, interstitial fibrosis and occasional crescent formation were significantly reduced in the E / N-IGF-3D cell aggregate treatment group.

[0194] Antibody deposition plays a key role in the pathogenesis of systemic lupus erythematosus. Fig.37 As shown, the deposition of C3 and IgG antibodies in the kidneys of MRL / Lpr mice transplanted with E / N-IGF-3D cell aggregates was significantly lower than that in the PBS-treated group and the TC-3D cell aggregate-treated group. These results indicate that in vivo treatment with E / N-IGF-3D cell aggregates is more effective in ameliorating the lupus-like phenotype of MRL / Lpr mice.

[0195] Analysis of immune cell subsets in spleen and perirenal lymph nodes of mice treated with 2.4E / N-IGF-3D cell aggregates

[0196] Compared with the PBS and TC-3D aggregates treatment groups, the Treg cell ratio in the spleen and lymph nodes was significantly increased in the E / N-IGF-3D cell aggregates treatment group. In addition to Treg cells, CD4+ effector T cells and CD11b+ cells secreting IFNγ and TNFα were significantly reduced in the E / N-IGF-3D cell aggregates treatment group, as shown in Figure 2. Fig.38 These results indicate that the E / N-IGF-3D cell aggregate treatment group can regulate immune cells, more effectively inhibit inflammation, and further improve MRL / Lpr renal injury.

[0197] In summary, the present invention provides a mesenchymal stem cell aggregate jointly empowered by a matrix and a cytokine, wherein the mesenchymal stem cell aggregate jointly empowered by a matrix and a cytokine is obtained by subculturing a novel mesenchymal stem cell in a 3D cell culture system, wherein the novel mesenchymal stem cell is obtained by subculturing a mesenchymal stem cell in a cadherin cell culture matrix and IGF-1, wherein the cadherin cell culture matrix is ​​prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc, and is pretreated with E / N-cad and 10 ng / mL IGF-1 to obtain an E / N-IGF-3D aggregate having a complete structure and clear boundaries. Compared with untreated TC-3D aggregates, the endogenous cadherin, IGF-1 receptor and its phosphorylated receptor of E / N-IGF-3D aggregate cells were significantly upregulated; anti-inflammatory gene expression was upregulated, including TSG-6 gene expression, which was upregulated by about 50%, IL-10 and HGF gene expression was also significantly upregulated (more than 4 times), and the cell metabolic pathway was more biomimetic to the MSC metabolic form in vivo, which is beneficial to improve the therapeutic effect after cell reinfusion; in vitro cell studies showed that E / N-IGF-3D aggregates can effectively inhibit the proliferation of activated T cells, efficiently reverse the fibrotic phenotype of skin fibroblasts, and promote the repair of damaged endothelial cells and improve the integrity of the vascular endothelium. The E / N-IGF-3D aggregates described in the present invention can treat systemic sclerosis. Compared with the TC-3D cell treatment group without pretreatment, the survival and anti-apoptosis ability of E / N-IGF-3D cells in an inflammatory environment are significantly improved, and the immune infiltration of lymphocytes and monocytes in the lesion site is effectively inhibited, the pro-inflammatory related CD4+ lymphocytes are reduced, and the anti-inflammatory Treg cells are increased; at the same time, the efficacy of E / N-IGF-3D aggregates in reversing fibrosis of the dermis and lung tissue is significantly improved; and E / N-IGF-3D aggregates significantly improve the vascular integrity and permeability of fibrotic lung tissue. The E / N-IGF-3D aggregates described in the present invention can treat Sjögren's disease. Compared with TC-3D, the E / N-IGF-3D aggregate treatment group significantly upregulated the salivary gland flow rate, improved the salivary gland / body weight ratio, reduced inflammatory cell infiltration, reduced acinar and duct atrophy, reduced salivary gland collagen deposition and fibrosis, and simultaneously increased the expression of aquaporins and ion transporters in damaged salivary glands, effectively improving the salivary gland function of Sjögren's disease.The E / N-IGF-3D cell aggregates described in the present invention are used to treat systemic lupus erythematosus mice. Compared with the TC-3D treatment group, the E / N-IGF-3D cell aggregate treatment group significantly inhibited the enlargement of perinephric lymph nodes and spleen, and inhibited lymphocyte proliferation; significantly reduced the physiological and biochemical indicators of systemic lupus erythematosus mice, including the protein, creatinine, urea nitrogen content in mouse urine, and various pathological parameters such as anti-dsDNA antibody levels, TNF-α and IgG concentrations in the blood; and the characteristics of the E / N-IGF-3D aggregate treatment group mice such as thickening of the glomerular mesangium and basement membrane, inflammatory cell infiltration, interstitial fibrosis and occasional crescent formation were significantly reduced; at the same time, the E / N-IGF-3D aggregates more effectively inhibited inflammation by regulating immune cells, further improving MRL / Lpr renal injury.

Claims

1. A mesenchymal stem cell aggregate empowered by matrix and cytokines, characterized in that: The aggregates are obtained by culturing mesenchymal stem cells in a culture medium supplemented with cell culture matrix and IGF-1 to obtain pretreated mesenchymal stem cells, and then continuing to culture them in a 3D cell culture system. The cell culture matrix is ​​prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc.

2. The mesenchymal stem cell aggregates jointly empowered by matrix and cytokines according to claim 1, characterized in that: The matrix and cytokine-combined mesenchymal stem cell aggregates are prepared by the following method: (1) diluting 1-3 portions of hE-cad-Fc and 1-3 portions of hN-cad-Fc to a total protein concentration of 1-30 μg / mL, adding to a tissue culture treated plate, incubating, and washing with PBS to obtain a cell culture matrix; (2) inoculating the mesenchymal stem cells into the cell culture matrix-enhanced cell culture plate obtained in step (1), and subculturing them to obtain matrix-enhanced mesenchymal stem cells; (3) culturing the matrix-enabled mesenchymal stem cells obtained in step (2) until the cells grow to 50-60% confluence, adding human recombinant IGF-1 to the culture medium, culturing until 70-80% confluence, digesting, and collecting the mesenchymal stem cells pretreated with cadherin combined with IGF-1; (4) The cells obtained in step (3) are added to a 3D cell culture system and cultured to obtain mesenchymal stem cell aggregates jointly empowered by matrix and cytokines.

3. The mesenchymal stem cell aggregates jointly empowered by matrix and cytokines according to claim 2, characterized in that: The incubation time in step (1) is 0.5-24 hrs, and the incubation temperature is 4-37°C.

4. The mesenchymal stem cell aggregates jointly empowered by matrix and cytokines according to claim 2, characterized in that: The concentration of IGF-1 added in step (3) is 0-15 ng / mL.

5. The mesenchymal stem cell aggregates jointly empowered by matrix and cytokines according to any one of claims 1 to 4, characterized in that: The cells and cell aggregates highly express endogenous cadherin and phosphorylated IGF-1 receptor.

6. The method for preparing mesenchymal stem cell aggregates pretreated with E-cadherin combined with IGF-1 according to claim 1, characterized in that: The matrix and cytokine-combined mesenchymal stem cell aggregates are prepared by the following method: (1) diluting 1-3 portions of hE-cad-Fc and 1-3 portions of hN-cad-Fc to a total protein concentration of 1-30 μg / mL, adding to a tissue culture treated plate, incubating, and washing with PBS to obtain a cell culture matrix; (2) inoculating the mesenchymal stem cells into the cell culture matrix-enhanced cell culture plate obtained in step (1), and subculturing them to obtain matrix-enhanced mesenchymal stem cells; (3) culturing the matrix-enabled mesenchymal stem cells obtained in step (2) until the cells grow to 50-60% confluence, adding human recombinant IGF-1 to the culture medium, culturing until 70-80% confluence, digesting, and collecting the mesenchymal stem cells pretreated with cadherin combined with IGF-1; (4) The cells obtained in step (3) are added to a 3D cell culture system and cultured to obtain mesenchymal stem cell aggregates jointly empowered by matrix and cytokines.

7. Use of the mesenchymal stem cell aggregates jointly empowered by matrix and cytokines as described in any one of claims 1 to 4 in the preparation of drugs for treating immune diseases.

8. The use according to claim 7, characterized in that The immune diseases include autoimmune diseases and inflammatory immune diseases.

9. The use according to claim 8, 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.

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