Use of a stromal-competent mesenchymal stem cell

Abstract

The present application relates to the field of biotechnology, in particular to the application of matrix-enabled mesenchymal stem cells, wherein the matrix-enabled mesenchymal stem cells are obtained by culturing mesenchymal stem cells on a cell culture matrix hENFc, and the cell culture matrix hENFc is prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc; the matrix-enabled mesenchymal stem cells can promote the nuclear transfer of beta-catenin, enhance the proliferation ability, stem maintenance ability, delay cell aging and apoptosis of in vitro expansion, the hMSCs cultured by long-term enabled culture (fifteenth generation and above) of hENFc do not have tumorigenicity, and have no adverse effects on animals, which meets the clinical application standard of hMSCs; can treat immune disorder diseases such as systemic lupus erythematosus, diabetic nephropathy, systemic sclerosis, Sjogren's syndrome, etc., and has a broad application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of stromal-primed mesenchymal stem cells. BACKGROUND

[0002] Immune diseases refer to the clinical symptoms caused by the damage or dysfunction of self-tissue cells due to the persistent and prolonged immune response of the immune system to self-antigens, which is caused by the breaking of the autoimmune tolerance state or the abnormality of autoimmune cell regulation of the body under the induction of internal and external factors such as genetic factors and environmental factors. Common immune diseases include systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome, hyperthyroidism, juvenile diabetes, diabetic nephropathy, primary thrombocytopenic purpura, autoimmune hemolytic anemia, lupus nephritis, rheumatoid arthritis, systemic vasculitis, ulcerative colitis, chronic hepatitis, and multiple neuritis. Immune diseases currently mainly rely on drug treatment, and common drugs include immunosuppressants, antibodies or blockers of anti-cytokine and its receptors, anti-immune cell surface molecule antibodies, and induction of immune tolerance by oral administration of self-antigens.

[0003] Stem cells have the characteristics of self-renewal and the ability to produce cells of different lineages, which have been widely explored in cell therapy for the treatment of various human diseases. Due to the regenerative properties of stem cells, the ability to secrete trophic factors and multilineage differentiation, and the absence of ethical restrictions, they have become an important source of stem cells in regenerative medicine. In addition, stem cells also exhibit low immunogenicity, homing effect and immunomodulatory function, and by affecting the microenvironment of damaged tissues, they enhance tissue repair and become the most common source of cells in preclinical and clinical studies. Currently, stem cells can treat a series of diseases by restoring organ homeostasis in inflamed, injured or diseased tissues.

[0004] Stem cell therapy for immune diseases has been applied in clinical practice, for example, Li Yuhua et al. found that adipose-derived mesenchymal stem cells can be used to treat autoimmune diseases in clinical practice [1] , and 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 themselves, the quality of MSCs decreases with the increase of in vitro culture time, because continuous subculture leads to changes in cell morphology, cell enlargement and eventually senescence; the homing effect of MSCs after in vitro expansion is significantly reduced, and the expression pattern changes significantly; traditional culture expansion gradually reduces the therapeutic efficacy of MSCs, which seriously affects the clinical application of MSCs. And it is difficult to predict the immune regulation and regenerative effects of MSCs in human trials.

[0005] Therefore, the mesenchymal stem cells which can promote the nuclear transfer of beta-catenin, enhance the proliferation ability of in vitro expansion, maintain the stemness, delay cell aging and apoptosis, have no tumorigenicity after being cultured by hENFc for a long time (eleventh generation and above), and have no any adverse effects on animals, meet the clinical application standards of hMSCs, become the technical problem to be solved by the person skilled in the art.

[0006] In order to solve the above technical problems, the application provides a new type of mesenchymal stem cells, the matrix-enabled mesenchymal stem cells are obtained by culturing 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. The matrix-enabled mesenchymal stem cells can promote the nuclear transfer of beta-catenin, enhance the proliferation ability of in vitro expansion, maintain the stemness, delay cell aging and apoptosis, have no tumorigenicity after being cultured by hENFc to the fifteenth generation, and have no any adverse effects on animals, meet the clinical application standards of hMSCs, and the matrix-enabled mesenchymal stem cells are used for treating immune diseases, and good treatment effects are obtained.

[0007] Reference:

[0008] [1] Li Y, Li Q, Xie Y. Application Progress of Adipose-derived Mesenchymal Stem Cells in Autoimmune Diseases [J]. Journal of Shanghai Jiaotong University (Medical Science), 2022, 42(08): 1131-1138.

[0009] [2] Shu Z, Yuan Y, Fu Q. Mechanism and Research Progress of Mesenchymal Stem Cell Therapy for Systemic Lupus Erythematosus [J]. Journal of Guangzhou Medical University, 2024, 52(02): 75-80. SUMMARY

[0010] The primary object of the application is to provide the application of the 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 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 to a total protein concentration of 1-30 μg / mL by hE-cad-Fc and hN-cad-Fc, and is added to a tissue culture treated plate; incubated for 0.5-24 hrs, and washed to obtain.

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

[0013] Preferably, the autoimmune disease includes one or more of systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome, hyperthyroidism, juvenile diabetes, diabetic nephropathy, idiopathic thrombocytopenia, autoimmune hemolytic anemia, the inflammatory immune disease includes one or more of lupus nephritis, rheumatoid arthritis, systemic vasculitis, ulcerative colitis, chronic hepatitis, polyneuritis.

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

[0015] Preferably, the mesenchymal stem cells are derived from a human, a pig or a mouse.

[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 application are: (1) the present application provides a use of matrix-enabled mesenchymal stem cells in the preparation of a drug for preventing and treating immune diseases, wherein the mesenchymal stem cells are enabled by cell culture matrix hENFc, the cell culture matrix hENFc is prepared from 1-3 parts of hN-cad-Fc and 1-3 parts of hE-cad-Fc, the matrix-enabled mesenchymal stem cells can promote the nuclear transfer of β-catenin, enhance the proliferation ability of in vitro expansion, maintain the ability of dryness, delay cell aging and apoptosis, hMSCs enabled by hENFc to the eleventh generation do not have tumorigenicity and have no adverse effects on animals, which meets the clinical application standard of hMSCs.

[0019] (2) 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, significantly inhibit PBMC proliferation Figure 5 A) and promote cell apoptosis Figure 5 B), MSC EN also inhibits the proportion of CD4+ and CD8+ T cell subsets, and improves the therapeutic effect of hMSCs in inflammatory and autoimmune diseases.

[0020] (3) the matrix-enabled mesenchymal stem cells can treat systemic lupus erythematosus, and the biochemical indicators and immune organ pathological indicators of the mice after treatment are significantly improved, the thickening of mesangial and basement membrane, inflammatory cell infiltration, interstitial fibrosis and occasional crescent formation are significantly reduced;

[0021] (4) The matrix-enabled mesenchymal stem cells can treat diabetic nephropathy, reduce the pathological symptoms of the kidney tissue of the mice after treatment, and significantly delay the progression of diabetic nephropathy; significantly inhibit the increase of blood glucose, 24-hour urinary protein, BUN and SCr of the rats; effectively improve the potential of hMSCs to slow down the kidney damage of diabetic rats, improve the kidney function of DKD rats, effectively reduce the cell damage caused by oxidative stress, alleviate the development of diabetic nephropathy, and have stronger anti-apoptosis ability.

[0022] (5) The matrix-enabled mesenchymal stem cells can inhibit the proliferation of activated T cells, and the MSCs EN The cells can inhibit the proliferation of activated T cells, and the MSCs

[0023] (6) The matrix-enabled mesenchymal stem cells can improve the function of the salivary glands of Sjogren's syndrome, up-regulate the flow rate of the salivary glands, reduce the infiltration of inflammatory cells, reduce the atrophy of acinar and duct, reduce the degree of fibrosis of the salivary glands, increase the expression of aquaporin, and improve the function of the salivary glands of Sjogren's syndrome. BRIEF DESCRIPTION OF DRAWINGS

[0024] The accompanying drawings illustrate the disclosed embodiments and serve to explain the principles of the disclosed embodiments. It should be understood, however, that the drawings are designed for purposes of illustration only and not as a definition of the limits of the application.

[0025] Figure 1 Preparation of hENFc

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

[0027] Figure 2 Characterization of hENFc

[0028] Note: (A) Surface elemental analysis of different substrates; (B) Stability test of different substrates; (C) Distribution and structure test of hE-cad-Fc and hN-cad-Fc on different substrates; (D) Hydrophilic and hydrophobic test of different substrates

[0029] Figure 3 Proliferation test of hMSCs on different substrates

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

[0031] Figure 4 Volume statistics of hMSCs on different substrates

[0032] Figure 5 Endogenous cadherin expression test of hMSCs on different substrates

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

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

[0035] Figure 7 OCT4 gene and protein expression of hMSCs on different substrates

[0036] Figure 8 Senescence test of hMSCs on different substrates

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

[0038] Figure 9 Apoptosis test of hMSCs on different substrates

[0039] Figure 10 Differentiation ability test of hMSCs on different substrates

[0040] 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

[0041] Figure 11 Carcinogenic and tumor suppressor gene expression dot plot of MSC EN

[0042] Figure 12 Karyotype analysis of MSC EN ​​

[0043] Figure 13 MSC EN tumorigenicity detection

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

[0045] Figure 14 Effect of IFN-γ stimulation on MSC EN viability

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

[0047] Figure 15 Effect of IFNγ stimulation on MSC EN anti-inflammatory capacity

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

[0049] Figure 16 Effect of MSC EN co-culture on PBMC proliferation and differentiation

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

[0051] Figure 17 Effect of MSC EN co-culture on CD4+T cell subtype in PBMC

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

[0053] Figure 18 Effect of MSC EN and PBMC co-culture system on inflammatory factors

[0054] Figure 19 Effect of MSC EN on mouse lupus nephritis

[0055] Figure 20 Effect of MSC ENEffects of transplantation on physiological and biochemical indexes of mice

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

[0057] Figure 21 Kidney pathological staining of mice 2 weeks after transplantation

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

[0059] Figure 22 MSC EN Kidney pathological score of mice 2 weeks after transplantation

[0060] Figure 23 Immunofluorescence staining of IgG and C3 in the kidney of mice 2 weeks after hMSCs transplantation

[0061] Figure 24 MSC EN Analysis of immune cell status in the perirenal lymph nodes of mice 2 weeks after transplantation

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

[0063] Figure 25 MSC EN Analysis of CD4+T cell subtypes in the perirenal lymph nodes of mice 2 weeks after transplantation

[0064] Figure 26 MSC EN Analysis of immune cell status in the spleen of mice 2 weeks after transplantation

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

[0066] Figure 27 MSC EN Analysis of CD4+T cell subtypes in the spleen of mice 2 weeks after transplantation

[0067] Figure 28 Construction of rat diabetic nephropathy model and MSC EN Treatment scheme

[0068] Figure 29 MSC EN Effects of transplantation on physiological and biochemical indexes of rats

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

[0070] Figure 30 MSC EN Effect of transplantation on rat kidney after 2 weeks

[0071] Note: (A) Kidney weight; (B) Kidney observation light microscope figure; (C) Kidney index

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

[0073] Figure 32 MSC EN Detection of oxidative stress level in rat kidney after 2 weeks of transplantation

[0074] Figure 33 MSC EN Effect of transplantation on rat kidney apoptosis after 2 weeks

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

[0076] Figure 34 MSC EN Western Blotting detection and quantitative statistics of apoptosis-related protein expression in rat kidney after 2 weeks of transplantation

[0077] Figure 35 MSC EN Effect of transplantation on rat systemic inflammation after 2 weeks

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

[0079] Figure 36 MSC EN Effect of transplantation on rat kidney function after 2 weeks Note: (A) Immunofluorescence staining of Synaptopodin protein; (B) Western Blotting detection and quantitative statistics of Nephrin, WT1 expression

[0080] Figure 37 MSC EN Cell inhibits PBMC proliferation

[0081] Figure 38 MSC EN Cell inhibits fibrosis

[0082] Figure 39 MSC EN Cell inhibits inflammatory infiltration

[0083] Figure 40 MSC EN Cell inhibits skin fibrosis

[0084] Figure 41 MSC EN Cell inhibits skin fibrosis

[0085] Figure 42 MSC EN Cell improves vascular permeability

[0086] Figure 43 MSC EN Therapeutic effect on Sjogren's syndrome

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

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

[0089] In the following examples, the reagents used are commercially available unless otherwise specified.

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

[0091] In the following examples, the hE-cad-Fc modified matrix is referred to as hEFc, and the hN-cad-Fc modified matrix is referred to as hNFc, wherein the hE-cad-Fc and hN-cad-Fc have been disclosed in the inventor's previous applications for 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", and the corresponding sequences are disclosed in detail, which will not be listed in this patent.

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

[0093] All data are presented as mean ± standard deviation; statistical differences between groups were assessed by one-way ANOVA. p < 0.05 was considered statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001, ns means no significant difference. All in vitro experimental results were from 3 independent parallel experiments, and all animal experimental results were from 5 independent parallel experiments, unless otherwise stated. All results were statistically analyzed using GraphPad Prism 8.0 software.

[0094] Example One, Preparation of hENFc matrix and matrix-enabled mesenchymal stem cells

[0095] 1. Preparation of hENFc matrix

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

[0097] 2. Adsorption amount-concentration and adsorption amount-time detection 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, 30 pg / mL, added to 96-well plates at 100 pL per well, and incubated at 37 °C for 2 hrs before further washing the plates 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 pg / mL, added to 96-well plates at 100 pL per well, and incubated at 37 °C for 0, 30, 60, 90, 120, 150, 180 min before further washing the plates 3 times with PBS. Finally, the modified surfaces were 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 non-specific interactions, then 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 3 times with PBS, the modified surfaces were incubated with HRP-labeled goat anti-rabbit / mouse IgG (H+L) antibody (1 :5000 dilution) for 2 hrs, 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 with a microplate reader.

[0099] 3. Hydrophilic / hydrophobic detection, elemental analysis and topography detection of hENFc substrates

[0100] The prepared TCPS, hEFc, hNFc and hENFc substrates were washed with ultrapure water for 5 times and air-dried, and the surface hydrophilicity of different surfaces was evaluated using a water contact angle (WCA) measuring instrument. The elemental changes before and after the modification of the substrate surface were evaluated using an X-ray photoelectron spectroscopy (XPS) instrument with a monochromatic Mg K a radiation source (1253.6 eV). C1s (binding energy 284.8 eV) 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 detection of hENFc substrates

[0102] Purified hE-cad-Fc and hN-cad-Fc were diluted in PBS to a total concentration of 5 μg / mL, added to 96-well plates at 100 μL per well, and incubated at 37°C for 2 hrs, followed by further washing of the plates 3 times with PBS; finally, the modified surfaces were assayed by Elisa, briefly, each substrate was incubated with 1.0% (w / v) BSA for 1 h to block nonspecific interactions, then incubated with goat anti-human IgG (H+L) antibody (1 :5000 dilution) for 2 hrs, followed by addition of TMB solution and incubation at 37°C in the dark; after 15 min, a stop buffer was added, and the absorbance was measured at 450 nm with a microplate reader.

[0103] 6. Results

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

[0105] As shown in Figure 1 A, the surface immobilization of hE-cad-Fc and hN-cad-Fc on TCPS was concentration-dependent, and the amount of surface protein immobilization was proportional to the concentration when the Cad-Fc concentration was lower than 5 μg / mL, and reached saturation when the Cad-Fc concentration was 10 μg / mL. Figure 2 B demonstrated that the amount of surface protein immobilization was also affected by the immobilization time, and the maximum amount of protein immobilization was achieved when the incubation time was 120 min. Figure 1 C showed that the fusion protein substrates with different ratios of hE-cad-Fc and hN-cad-Fc could be prepared by controlling the Cad-Fc concentration and immobilization time. To avoid the interaction of the two Cad-Fc in solution, I first observed the minimum concentration of the two Cad-Fc interaction by micro-thermal migration experiment, which was 400 μg / mL, much higher than the protein solution concentration of the saturated immobilization amount (10 μg / mL). To construct the cadherin fusion protein substrates with different subtypes and ratios, we kept the total concentration of hE-cad-Fc and hN-cad-Fc constant at 5 μg / mL, while changing their ratios (hE-cad-Fc:hN-cad-Fc = 2:1, 1:1 and 1:2 to prepare hEFc, hNFc and hENFc substrates with different Cad-Fc ratios Figure 1 D).

[0106] 6.2 Physicochemical characterization of hENFc substrates

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

[0108] The inventors made the influence of hENFc substrate on the cell adhesion and proliferation of hMSCs. With the increase of the proportion of hE-cad-Fc in the hN-cad-Fc fusion protein substrate, the expression of OCT4 of hMSCs increased. When the proportion of hE-cad-Fc was greater than or equal to hN-cad-Fc, the expression level of OCT4 gene of hMSCs was significantly higher than that of hMSCs cultured on TCPS surface, and the expression level no longer increased with the increase of the proportion of hE-cad-Fc. Therefore, the hENFc complex substrate (hE-cad-Fc:hN-cad-Fc=1:1) was selected as the preferred substrate for subsequent research.

[0109] Example Two, Preparation and Function of hENFc Substrate Enabled hMSCs

[0110] 1. Preparation of hENFc Substrate Enabled hMSCs

[0111] First, hMSCs were cultured on TCPS to the 2nd generation; then hMSCs were inoculated into hEFc, hNFc or hENFc substrate T-75 cell culture bottles at 1x10 6 cells per bottle, and continuously cultured to the 15th generation under conventional expansion conditions to obtain hMSCs of different generations.

[0112] The following experiments used unmodified TCPS substrate to prepare hMSCs of each generation as a control. hMSCs enabled on hEFc substrate were referred to as MSC E , hMSCs enabled on hNFc substrate were referred to as MSC N , hMSCs enabled on hENFc substrate were referred to as MSC EN , and hMSCs normally cultured on TCPS substrate were referred to as MSC T .

[0113] 2. Detection of the Proliferation Ability of hENFc Substrate Enabled hMSCs

[0114] ​All cells on different substrates were collected at the end of each passage and counted after excluding dead cells using trypan blue. Alternatively, MSCs were seeded onto TCPS, hEFc, hNFc or hENFc substrates and incubated for 24, 48 hrs before cell viability was determined using CCK-8. To compare the cell proliferation rate at different passages, the doubling time (DT) and population doubling level (PDL) were calculated according to the following formula:

[0115]

[0116] where N(t) is the cell number at passage, N(to) is the cell number seeded at the previous passage, 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 onto six-well TCPS, hEFc, hNFc or hENFc substrate surfaces at a density of 1 x 104cells / cm2and incubated for 2 hrs before cell membrane and nuclear proteins were extracted: the cells were digested with EDTA, the precipitate was collected by centrifugation at 500 x g for 3 min, 200 μL of a protein extraction reagent was added to each sample and vortexed at high speed for 15 sec, the sample was then incubated on ice for 10 min and centrifuged at 4°C at 12,000 x g for 10 min, 100 μL of a nuclear protein extraction reagent was then added, vortexed for 15 sec and incubated on ice for 10 min, and finally centrifuged at 4°C at 16,000 x g for 10 min to obtain the nuclear protein; the expression and distribution of β-catenin protein were detected by Western Blotting.

[0119] 4. Detection of stemness of hENFc substrate-enabled hMSCs

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

[0121] PCR was performed to detect the expression of relevant genes using the following primers:

[0122]

[0123] 5. Detection of the cycle of hENFc substrate-enabled hMSCs

[0124] At passages 6, 11 and 15, 1 x 10 6 MSCs T and MSCs EN were harvested and fixed in 70% cold ethanol for 2h to overnight; after PBS washing for 3 times, cell pellets were obtained by centrifugation at 1000rpm for 10min at room temperature; 100μL RNase A solution was added to each sample, and the cells were resuspended and incubated at 37°C for 30min; 400μL PI staining solution was added and mixed well, and the cells were incubated at 4°C for 30min in the dark; the red fluorescence at 488nm excitation wavelength was recorded by flow cytometry.

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

[0126] For senescence-associated β-galactosidase assay, the β-galactosidase activity in MSCs T and MSCs EN was detected at passages 6, 11 and 15 using SA-β-gal staining kit. Briefly, the cells were cultured in modified or unmodified 6-well plates to 80% confluence, and then fixed with 4% formaldehyde for 15min after PBS washing; subsequently, the cells were incubated with freshly prepared β-galactosidase staining solution at 37°C overnight in a CO2-free incubator; after PBS washing for 3 times, the cells were photographed using an optical microscope, and the number of β-galactosidase positive stained cells was counted per 200 cells in randomly selected fields.

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

[0128] At passages 6, 11 and 15, 1 x 10 6 MSCs T and MSCs EN were harvested by trypsin without EDTA; the cells were resuspended with 1ml deionized water diluted Binding Buffer (diluted at 1:9); 5μL Annexin V / FITC was added to each 100μL cell suspension, and the mixture was incubated at room temperature in the dark for 5min; after adding 5μL propidium iodide solution and 400μL PBS, the mixture was placed in a 5ml flow tube, and immediately subjected to flow cytometry detection. In addition, the cells without staining were used as blank tubes; the cells stained with Annexin V / FITC only were used as single staining tubes.

[0129] 8. Differentiation potential detection of hENFc matrix-primed hMSCs

[0130] The 6th passage MSCs T , 11th passage MSCs T and 11th passage MSCs ENCells were cultured in differentiation medium for 14d to test the tri-lineage differentiation ability of hMSCs. For osteogenic differentiation: osteogenesis was induced by culturing cells in DF12 supplemented with 50 pg / mL ascorbic acid 2-phosphate, 10 nM dexamethasone (Dex), 10 mM beta-glycerophosphate and 10% FBS, 1% penicillin-streptomycin, and the 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 pg / mL ascorbic acid 2-phosphate, 1 mM Dex, 0.1 mM indomethacin and 4 U / L insulin, and the mature cells were detected using Oil Red O staining kit; for chondrogenic differentiation: chondrogenesis was induced by culturing cells in DF12 supplemented with 2% FBS, 50 pg / mL ascorbic acid 2-phosphate, 50 pg / mL L-proline, 100 nM Dex, 10 ng / mL TGF-β1, and 1% human insulin transferrin and sodium selenite, and the mature cells were detected using Toluidine Blue Cartilage Solution.

[0131] PCR was used to detect the expression of three-phase differentiation-related genes using the following primers:

[0132]

[0133]

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

[0135] To examine the tumorigenicity of hENFc-primed cultured hMSCs, 1 x 10 7 passage 6 MSCs T (n=3) and passage 11 MSCs EN (n=6) were subcutaneously transplanted into the abdomen of 4-week-old NOD-SCID mice; the body weight of the mice was measured every 3 days, and the volume of the tumor was measured according to the following formula, and the mice were sacrificed after continuous feeding for seven weeks. Mice injected with 1 x 10 7 personal glioblastoma (U87) (n=3) were used as positive controls, and mice injected with an equal volume of PBS (n=3) were used as negative controls.

[0136]

[0137] 10. Results

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

[0139] Although the proliferation ability of all cells showed a gradually weakened trend with the passage of cells, the number of cells harvested at each passage also gradually decreased Figure 3). However, the proliferation ability of hMSCs cultured on TCPS surface (MSC T ) was well maintained, especially for hMSCs cultured on hENFc matrix (MSC EN ), the cell doubling time was always kept within 25 hrs until the eleventh passage, and the proliferation ability did not change significantly. It suggested that the hENFc matrix effectively maintained the in vitro proliferation activity of hMSCs by mimicking the E-cadherin and N-cadherin mediated cell-cell interaction. And the size of hMSCs cultured on hENFc matrix did not change with the passage amplification, the volume of MSC EN was always within 2000 μm 3 (about 16 μm in diameter) Figure 4 ).

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

[0141] As shown in Figure 5 , the in vitro cultured hMSCs lost the expression of E-cadherin, although the expression of N-cadherin was retained, but also gradually lost with the culture. While cultured on the hENFc matrix, the hMSCs were re-endowed with the expression of endogenous E-cadherin and maintained the presence of N-cadherin. The expression of membrane cadherin plays an important role in cadherin-catenin complex, and the combination of hE-cad-Fc and hN-cad-Fc matrix (mass ratio of 1:1) promoted the nuclear translocation of β-catenin Figure 6 .

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

[0143] E-cadherin plays a key role in stem cell stemness maintenance, we continued to investigate the expression of OCT4, a stemness index of hMSCs in long-term passage. The results of gene and protein detection showed that compared with MSC T , the stemness of MSC EN was maintained in continuous passage, and the stemness of MSC EN of the sixth, eleventh and fifteenth passages were all higher than that of MSC T . What is particularly important is that the stemness of MSC EN of the eleventh passage was significantly better than that of MSC T of the sixth passage Figure 7 .

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

[0145] Replicative senescence is the biggest obstacle to the in vitro culture of hMSCs, leading to loss of cell proliferation capacity and alterations in secretory profiles, thus limiting the therapeutic efficacy of the cells. Cell cycle analysis revealed that MSCs at different passages... EN The number of cells in S phase was significantly higher than that in MSCs. T β-galactosidase staining further confirmed the presence of MSCs. EN The percentage of senescent cells was consistently below 10% and significantly lower than that of MSCs. T ( Figure 8 The result is as follows: Figure 9 As shown, after long-term in vitro culture, MSCs T A large number of apoptosis occurred, while MSCs EN The proportion of apoptotic cells did not change significantly with increasing passage number, making the eleventh generation MSCs... EN In terms of the ratio of senescent and apoptotic cells, compared with sixth-generation MSCs T Basically the same.

[0146] We further examined the differentiation capacity of each generation of hMSCs, comparing it with that of the eleventh generation MSCs. T (MSC T Compared to -P11), in the eleventh generation MSC EN (MSC EN -P11) and the sixth generation MSC EN (MSC T More significant Oil Red O-positive lipid droplets and expression of PPAR-γ, CEBPA, and LPL genes were found in P6. Figure 10 A), MSC EN -P11 and MSC T There was no statistically significant difference between -P6. This demonstrates that hENFc matrix-encapsulated MSCs... EN -P11 and conventionally cultured MSCs T -P6 exhibits the same adipogenic differentiation characteristics. Similarly, in MSCs EN Alizarin Red S and Toluidine Blue positive cells were also detected in cells after P11 directed differentiation. Quantitative real-time PCR detection of mature bone and cartilage markers also confirmed the presence of MSCs. EN -P11 and MSC T -P6 has the same osteogenic ( Figure 10 B) and cartilage ( Figure 10 C) The potential for differentiation.

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

[0148] In vitro expansion and culture of MSCs inevitably leads to replicative senescence, resulting in genomic instability, a serious drawback hindering the development of MSC-based cell therapy. Therefore, we examined MSCs cultured to the eleventh generation using hENFc matrix empowerment. EN Compared with conventional culture to the sixth generation of MSCs T The expression of genes related to genome stability and chromosome karyotype were analyzed to investigate potential genetic instability. Analysis of gene expression levels related to genome stability transformation in transcriptome sequencing data showed that ( Figure 11 ), eleventh generation MSC EN MSCs cultured normally at the sixth generation T There were no statistically significant differences in the expression of proto-oncogenes and tumor suppressor genes among cells, and the expression levels of c-Myc, P21, P53, and P16 genes did not change significantly. Karyotype analysis did not reveal any clones with chromosomal abnormalities; no chromosomal translocations, deletions, or changes in chromosome number were observed. hENFc-empowered hMSCs maintained chromosomal stability. Figure 12 ).

[0149] Tumorigenicity is another risk factor to consider in the clinical application of MSCs. Compared to iPSCs and ESCs, although MSCs themselves do not possess tumorigenicity, have low genomic instability, and a low likelihood of forming teratomas, to ensure the safety of long-term hENFc matrix-encapsulated hMSCs in clinical applications, we will use MSCs that meet clinical criteria... T hENFc-enhanced MSCs cultured to the eleventh generation EN Human astrocytoma cells and U87 cells were injected subcutaneously into nude mice. Seven weeks later, the potential for these cells to form tumors was investigated. Figure 13 As shown in Figure A, mice inoculated with U87 developed a pea-sized lump at the injection site, while those inoculated with MSCs... T and MSC EN No tumors formed in any of the nude mice. To monitor the overall health of the mice, we measured their weight, and the results showed that mice inoculated with PBS and MSCs showed no abnormalities. T MSC EN There was no statistically significant difference in body weight between the mice and the U87 mice. Figure 13 B). MSC vaccination T and MSC EN No bulge formed at the injection site in the mice afterward. Figure 13 C) These results indicate that hMSCs cultured to the eleventh generation with hENFc empowerment do not have tumorigenicity and have no adverse effects on animals, meeting the standards for clinical application of hMSCs.

[0150] Example 3, MSCEN Applications in anti-inflammatory and immunomodulatory functions

[0151] 1. Experimental Methods

[0152] 1.1 Detection of inflammatory response in hENFc matrix-empowered hMSCs

[0153] Both MSCT and MSCEN were performed at a rate of 1×10⁻⁶ per square centimeter. 4 Cells were seeded at a density of 1,000 cells per well in 96-well TCPS plates 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 assessed as follows: MSCs were seeded into 96-well TCPS, hEFc, hNFc, or hENFc substrates and incubated for 4, 24, and 48 hours. After incubation, the supernatant was removed, and the cells were washed three times with PBS. 100 μL of CCK-8 (1:10 dilution) was added to each well, and the cells were incubated at 37°C for 4 hours. The absorbance was measured at 450 nm using a microplate reader.

[0155] Both MSCT and MSCEN were performed at a rate of 1×10⁻⁶ per square centimeter. 4 Cells were seeded at a density of 1000 cells per well in a 6-well TCPS plate and cultured for 24 hours. Then, IFN-γ was added to the culture medium at a final concentration of 10 ng / mL and cultured for another 24 hours. The supernatant and cells were then collected for further analysis.

[0156] The content of soluble tumor necrosis factor receptor 1 (sTNFR1) in the supernatant was detected by ELISA. First, all supernatant was centrifuged at 300×g for 10 min to remove precipitate; 300 μL of diluted washing buffer (1:20 dilution) was added and the plate was soaked for 30 sec; all washing buffer was discarded and the plate was patted dry on absorbent paper; 100 μL of culture medium or cell culture supernatant sample or diluted standard was added to each well; 50 μL of detection antibody (1:100 dilution) was added to each well; the plate was incubated at room temperature for 2 hours; the liquid was discarded, and 300 μL of washing buffer was added to each well to wash the plate 6 times, discarding the washing buffer after each wash and patting the plate dry; 100 μL of... Horseradish peroxidase-labeled streptavidin (1:100 dilution) was incubated at room temperature for 45 min. The liquid was discarded, and 300 μL of washing buffer was added to each well to wash the plate 6 times, discarding the washing buffer after each wash and patting the plate dry. 100 μL of chromogenic substrate TMB was added to each well, and the plate was incubated at room temperature in the dark for 30 min. 100 μL of stop solution was added to each well; at this point, the color changed from blue to yellow. Within 30 min, dual-wavelength detection was performed using a microplate reader to measure the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 570 nm, and the concentrations were calculated. 1.2 MSC ENCo-culture experiment with peripheral blood mononuclear cells

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

[0158] For the PBMC proliferation test, the isolated PBMC suspension was centrifuged at 300 x g for 10 min, and all the supernatant was discarded; 1 ml of PBS containing 0.1% FBS was added to resuspend the cells, 2 μM CFSE was added, and after being mixed well, the cells were cultured at 37°C and 5% CO2 for 10 min; 3 times the volume of pre-cooled RPMI1640 medium was added, and the cells were terminated at 4°C for 5 min; the cells were centrifuged at 300 x g for 10 min, the supernatant was discarded, 1 ml of PBS containing 0.1% FBS was added to resuspend the cells, the cells were centrifuged at 300 x g for 10 min, the supernatant was discarded, and the above steps were repeated once, and then the obtained cells were used to carry out the co-culture experiment; after the co-culture was completed, the cells in the culture medium were collected and analyzed by flow cytometry.

[0159] For the PBMC apoptosis detection, the following steps were performed: at the 6th, 11th and 15th passages, 1 x 10 6 MSC T and MSC EN Cells; the cells were resuspended with 1 ml of Binding Buffer diluted with deionized water (diluted at 1:9); 5 μL of AnnexinV / FITC was added to each 100 μL of cell suspension and mixed well; the cells were incubated at room temperature for 5 min in the dark; after adding 5 μL of propidium iodide solution and 400 μL of PBS, the cells were placed in a 5 ml flow tube, and flow cytometry detection was immediately performed. In addition, unstained cells were used as blank tubes; cells stained with AnnexinV / FITC only were used as single-stained tubes.

[0160] For T lymphocyte and T lymphocyte subsets determination in PBMC cells, 1 ml PBS was added to resuspend the cells obtained after the above co-culture, the cells were centrifuged at 300 x g for 10 min, the supernatant was discarded, and the PBMC subsets were characterized by using the following antibodies: CD4+T cells (FITC-labeled anti-human CD4 antibody, 1:500 dilution), CD8+T cells (FITC-labeled anti-human CD8 antibody, 1:500 dilution), Th1 / Th2 (FITC-labeled anti-human CD4, PE-labeled anti-human IL4, and APC-labeled anti-human IFN-γ antibodies, 1:500 dilution), Treg (FITC-labeled anti-human CD4, PE-labeled anti-human Foxp3, and APC-labeled anti-human CD25 antibodies, 1:500 dilution), and Th17 (FITC-labeled anti-human CD4 and APC-labeled anti-human IL17A antibodies, 1:500 dilution). After the addition of the antibodies, the cells were incubated at 4°C in the dark for 30 min. It should be noted that, for Foxp3 staining, 750 μL of a membrane-breaking agent was first added, and the cells were incubated at 4°C in the dark for 60 min. Then, 1 ml of PBS was added to wash the cells once, the cells were centrifuged at 300 x g for 10 min, the supernatant was discarded, 500 μL of PBS was added to resuspend the cells, the cells were filtered, and the cells were analyzed by flow cytometry.

[0161] For the detection of the secretion amounts 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 by the Elisa experiment as described above.

[0162] 2. Results

[0163] 2.1 Influence of hENFc matrix conditioning on the inflammatory environment response ability of hMSCs

[0164] The increase of inflammatory mediators can affect the activity of MSCs and the expression of immunomodulatory factors. In order to characterize the response of hMSCs to inflammatory environment stimulation, we stimulated MSCs with gradient concentrations of IFN-γ (0-30 ng / mL) T and MSCs EN After 24 hrs, the viability and factor expression of the cells were characterized. Figure 14 It was shown that, with the increase of the concentration of IFN-γ, the cell viability of hMSCs was significantly reduced, and when the concentration of IFN-γ was greater than 15 ng / mL, the viability of MSCs EN was significantly higher than that of MSCs T The stimulation of IFN-γ can up-regulate the expression of pro-apoptotic protein Bax and inhibit the expression of anti-apoptotic protein Bcl-2, further activate Caspase3 to execute cell apoptosis, but the anti-apoptotic ability of MSCs EN was significantly better than that of MSCs TIn a non-inflammatory environment, hMSCs basically do not express the anti-inflammatory factor IDO, but upon IFN-γ stimulation, they immediately express high levels of IDO, while MSCs... EN MSCs exhibit a stronger responsiveness, with IDO showing twice the gene and protein expression of MSCT. Furthermore, regardless of IFN-γ stimulation, MSCs... EN It has a stronger ability to release sTNFR1. Figure 15 These results indicate that hENFc matrix-empowered hMSCs not only have better survival capabilities in the lesion microenvironment but also exhibit stronger environmental awareness and anti-inflammatory abilities. The IDO primer sequences are as follows:

[0165]

[0166] 2.2 MSC EN Effects on the proliferation and differentiation of peripheral blood mononuclear cells

[0167] In immunology, immune responses are widely assessed using the reactions of PBMCs. Based on the ability of MSCs to influence PBMC proliferation, apoptosis, and differentiation in co-culture systems, we evaluated MSCs. EN Immunomodulatory activity. Flow cytometry analysis showed that, compared with untreated and MSCs... T In comparison, MSC EN It can significantly inhibit PBMC proliferation ( Figure 16 A) and promoting apoptosis ( Figure 16 B), MSC EN It also suppressed the ratio of CD4+ and CD8+ T cell subsets ( Figure 16 C and Figure 16 D)

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

[0169] likeFigure 18 As shown, in the MSC-PBMC co-culture group, the levels of proinflammatory factors TNF-a, IFN-g and IL-1 b were reduced, and the level of anti-inflammatory factor TGF-b was increased. Importantly, compared with MSC EN The changes in the levels of IFN-g and IL-1 b in the co-culture system were greater. It is generally believed that immune balance, especially the balance between proinflammatory factors and immunosuppressive cytokines, is crucial for maintaining human balance. These results show that the enabled culture of hENFc can improve the therapeutic effect of hMSCs in inflammatory and autoimmune diseases.

[0170] Example Four, MSC EN Treatment of systemic lupus erythematosus

[0171] 1. Experimental Methods

[0172] 1.1 Modeling of Systemic Lupus Erythematosus Nephritis

[0173] The model of systemic lupus erythematosus (SLE) provides a valuable model for evaluating the immunomodulatory and therapeutic potential of MSCs due to its autoimmune nature, multi-organ involvement, and similarity to the pathology of human systemic lupus erythematosus. We established a model of systemic lupus erythematosus using MRL / lpr mice Figure 19 At the end of the two-week transplantation period, it was observed that the transplanted MSCs EN The mice in the treatment group showed a significant improvement in lupus symptoms, in stark contrast to the PBS treatment group and the MSC T treatment group.

[0174] 1.2 MSC EN Transplantation

[0175] Eighteen 8-week-old female MRL / lpr lupus mice were raised in a SPF environment for 2 weeks; the treatment groups were injected with 1 x 10 6 MSC T , MSC EN , and the control group was injected with 200 μL of normal saline; the mice were weighed weekly; all animals were raised and experiments were strictly in accordance with the animal care and use guidelines approved by Gulou Hospital of Nanjing University, and appropriate measures were taken to ensure the minimum pain and discomfort of the animals. At 12 weeks, the mice were anesthetized by intraperitoneal injection, and blood was collected from the eyeball, and the mice were sacrificed by cervical dislocation, the spleen and lymph nodes were weighed, and part of the kidney was immediately placed in liquid nitrogen for storage.

[0176] Part of the kidney put into formaldehyde fixed overnight, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol in low concentration to high concentration in turn processing specimens 2 hrs, anhydrous ethanol dehydration 2 hrs, repeated 1 times, and then the tissue block is placed in xylene transparent, after transparent samples are placed in liquid paraffin, after the paraffin completely immersed in the tissue block, embedding, cooling fixed into a block; the embedded sample on the paraffin microtome section, then placed in warm water to expand the section, carefully paste the expanded paraffin thin section to the glass slide, then 45 DEG C constant temperature drying paraffin patch, room temperature preservation.

[0177] Part of the kidney put into formaldehyde fixed overnight, 30% sucrose solution dehydration to kidney bottom; remove the tissue, pre-cooled PBS washing 2 times, then add appropriate amount of OCT embedding agent to immerse the tissue; pre-cooled OCT is fixed on the microtome, cut into thin section, paste to the glass slide and store in-80 DEG C refrigerator for standby.

[0178] 1.3 mouse urine protein detection

[0179] The mouse urine is collected by metabolic cage, and the mouse urine to be tested is diluted to a suitable concentration, so that the total volume of the sample is diluted to 5 μL, 195 μL of coomassie brilliant blue G250 solution is added, and the mixture is mixed thoroughly and stands for 2 min; the absorbance at 595 nm is measured, and the urine protein concentration is calculated.

[0180] 1.4 mouse serum ds-DNA, TNF-α detection

[0181] After the mouse angular vein blood is taken, it is placed in a 1.5 mL centrifuge tube, centrifuged at 300 x g for 10 min, the supernatant is transferred to a new 1.5 mL centrifuge tube, and stands for 30 min; the supernatant is serum, which is transferred to a new centrifuge tube and stored in a-80 DEG C refrigerator for standby. Elisa experiment is used to analyze the content of ds-DNA and TNF-α in mouse serum.

[0182] 1.5 mouse kidney pathological staining

[0183] The paraffin section is deparaffinated and hydrated by using 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: the treated section is placed into a hematoxylin staining box and dyed for 5 min; the floating color is washed in water, and the section is gently washed with running water for 10 min; the section is differentiated with hydrochloric acid alcohol for 1 sec; the floating color is washed in water, and the section is washed with running water for 8 min; the section is dyed with eosin for 90 sec; the floating color is washed in water, and the section is gently washed with running water for 10 min.

[0185] ②For PAS staining: Put the treated sections into the periodic acid solution, and oxidize at room temperature for 25 min; rinse with running water for 2 times, 2 min each time, and gently rinse with distilled water for 2 times, 2 min each time; immerse in Schiff Reagent at 37°C for 10 min in the dark; wash with sodium sulfite solution for 2 times, 2 min each time; rinse with running water for 2 times, 2 min each time, and gently rinse with distilled water for 2 times, 2 min each time; immerse in hematoxylin staining box for 5 min; wash off the floating color in water, and gently rinse with running water for 10 min.

[0186] ③For Masson staining: Put the treated sections into Weigert iron hematoxylin staining solution for 10 min; rinse with running water for 10 min; return blue with Masson blue solution for 5 min; rinse with running water for 10 min, and rinse with distilled water for 2 times, 2 min each time; immerse in ponceau red staining solution for 10 min; wash with weak acid working solution (distilled water: weak acid solution = 2:1) for 1 min; wash with 1% phosphomolybdate solution for 2 min; wash with weak acid working solution for 1 min; directly immerse in aniline blue staining solution for 2 min; wash with weak acid working solution for 1 min.

[0187] After all the sample staining is completed, dehydrate according to 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; dry in a ventilated place, immerse in xylene for 5 min; immerse in fresh xylene for 5 min; dry in a ventilated kitchen, and then embed with neutral resin, and store at room temperature.

[0188] 1.6 Mouse kidney histopathological score

[0189] According to the research of Tao et al., the H&E, PAS and Masson staining results of the sections are scored according to the degree of inflammatory cell infiltration and mesangial proliferation in glomeruli, renal interstitium and blood vessels:

[0190] Table 3.3 Kidney pathological score

[0191]

[0192] 1.7 Mouse kidney immunofluorescence staining

[0193] Rinse the frozen sections with running water for 5 min to remove OCT, and perform immunofluorescence staining to detect the expression and distribution of IgG and C3 in different kidney tissues.

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

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

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

[0197] For T cell proliferation and differentiation staining, 1 x 10 6 cells were placed in a 96-well U-shaped plate, 5 μL of count beads was added to each well, and centrifuged at 2000 rpm for 5 min; BV421-labeled anti-mouse TCR beta antibody (1:500 dilution), FITC-labeled anti-mouse CD4 antibody (1:500 dilution), BV510-labeled anti-mouse CD8 antibody (1:500 dilution), BV605-labeled anti-mouse CD62L antibody (1:500 dilution), BV711-labeled anti-mouse CD44 antibody (1:500 dilution), APC-labeled anti-mouse CD25 antibody (1:500 dilution), Percp Cy5.5-labeled anti-mouse CD69 antibody (1:500 dilution), PE Cy7-labeled anti-mouse Ki67 antibody (1:500 dilution), PE-labeled anti-mouse Foxp3 antibody (1:500 dilution), and APC Cy7(780)-labeled anti-mouse live dye antibody (1:500 dilution) were added, and incubated at 4°C in the dark for 30 min. Note that for intranuclear antibody staining, 750 μL of membrane breaker was added first, and incubated at 4°C in the dark for 60 min; wash the cells with 1 ml of PBS containing 2% FBS once, centrifuge at 300 x g for 10 min, and discard the supernatant; resuspend with 150 μL of PBS containing 2% FBS, filter, and analyze the cells by flow cytometry.

[0198] For T cell subpopulation staining, 2 x 10 5Cells were resuspended in 200 uL of 1640 complete medium containing PMA, INO and BFA (1:1000 dilution) in 96-well U-bottom plates. After mixing, the cells were incubated at 37°C for 6 h. Then, the cells were centrifuged at 2000 rpm for 5 min. After adding 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-labeled anti-mouse IFN-γ antibody (1:500 dilution), PE-labeled anti-mouse IL4 antibody (1:500 dilution), and BV650-labeled anti-mouse TNF-α antibody (1:500 dilution), the cells were incubated at 4°C for 30 min in the dark. For intranuclear antibody staining, 750 uL of membrane breaker was first added to the cells, which were then incubated at 4°C for 60 min in the dark. Then, the cells were washed once with 1 mL of PBS containing 2% FBS, centrifuged at 300 x g for 10 min, and resuspended with 150 uL of PBS containing 2% FBS. After filtration, the cells were analyzed by flow cytometry.

[0199] 2. Results

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

[0201] Body weight loss, increased proteinuria, increased anti-dsDNA antibody levels, and TNF-α expression are the most commonly used indicators for clinically diagnosing SLE. To study the disease state of MRL / lpr mice after MSC transplantation, the body weight, protein content in urine, and anti-ds-DNA antibody and TNF-α concentrations in the blood of mice were detected by Elisa kit. It was found that, compared with the PBS treatment group and the MSC T treatment group, the MSC EN treatment group had more normal physiological and biochemical indicators. Figure 20 ).

[0202] 2.2 Effect of MSC EN transplantation on kidney histopathology and antibody deposition

[0203] We next tested whether MSC EN treatment in vivo could have beneficial effects on the renal histopathology and autoantibody production of SLE mice. The results of the renal histopathology of MRL / lpr mice were observed and analyzed using H&E, PAS, and Masson pathological staining. It was found that the mice treated with hMSCs showed a reversal of glomerular injury. In addition, Masson staining, as well as H&E and PAS staining, showed that, compared with the PBS treatment group and the MSC T treatment group, the MSC ENThe treated mice also showed a significant reduction in glomerular, interstitial, and perivascular damage. Figure 21 It is worth noting that, compared with the PBS treatment group and MSC... T Compared to the treatment group, MSC EN The treatment group mice showed a significant reduction in features such as glomerular mesangial and basement membrane thickening, inflammatory cell infiltration, interstitial fibrosis, and occasional crescent formation. Figure 22 ).

[0204] Antibody deposition plays a crucial role in the pathogenesis of lymphoma (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 group. T Treatment group ( Figure 23 These results indicate that MSC EN In vivo treatment improved the lupus-like phenotype in MRL / lpr mice.

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

[0206] The results are as follows Figure 24 The display shows that MSC EN Treatment reduced the number of CD4+ and CD8+ T cells in the perirenal lymph nodes, inhibiting their activation (CD69+), proliferation (Ki67+), and effector function (CD44+CD62L-), while increasing the proportion of unsensitized T cells (CD44-CD62L+). We further analyzed the proportions of Th1 cells, Th2 cells, and Treg cells in the mouse perirenal lymph nodes to determine MSCs. EN The effects of treatment on various T helper cell subsets. Results are as follows: Figure 25 The results showed that treatment with hMSCs led to 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). These changes occurred in MSCs. EN This was more pronounced in the treatment group. Compared to the PBS treatment group and MSC... T Compared to the treatment group, MSC EN The Th1 / Th2 ratio in the perirenal lymph nodes of treated mice also decreased, indicating that in MSCs EN Th2 cells were dominant in the perirenal lymph nodes of treated mice.

[0207] We also observed similar results in the spleen. Figure 26 and Figure 27 These findings are consistent with MSC EN Compared to MSC TCorresponding to the stronger ability to modulate T cell subsets and promote anti-inflammatory environment. These results demonstrate that MSCs EN induced T cell responses and Th cell subset balance play an important role in LN, highlighting the advantages of hENFc matrix-enabled culture in enhancing the therapeutic effect of hMSCs.

[0208] Example Five, MSCs EN Treatment of diabetic nephropathy in rats

[0209] 1. Experimental Methods

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

[0211] All 30 SD rats were adaptively fed for 1 week, after which they were given a high-fat diet (n = 20) and a normal diet (n = 10) for 8 weeks, respectively, to ensure that the rats had normal blood glucose (blood glucose < 250 mg / dL); after eight weeks, all rats were fasted for 12 h, and the high-fat diet-fed rats were injected intraperitoneally with STZ dissolved in sodium citrate solution at 35 mg / kg, while the normal diet-fed rats were injected intraperitoneally with an equal amount of sodium citrate solution. Blood glucose was continuously monitored for 3 days, and when the blood glucose concentration of the high-fat diet-fed rats plus STZ injection group was ≥ 250 mg / dL, it was considered that a type 2 diabetic model had been successfully constructed. Figure 28

[0212] After the type 2 diabetic model was successfully constructed, the rats were gradually transitioned to a normal diet, and the rats' state was observed and the rats' blood and urine indicators were detected; after 2 weeks of continuous culture, the rats' 24 h urine was collected, and when the urine protein was > 20 mg / 24 h and significantly higher than that of the normal control group, it was considered that kidney damage had occurred at that time, and a diabetic kidney disease (DKD) rat model had been successfully constructed. All animals were housed and experiments were strictly in accordance with the guidelines for animal care and use approved by Nanjing University Drum Tower Hospital, and appropriate measures were taken to ensure the minimum pain and discomfort of the animals.

[0213] 1.2 MSCs EN Transplantation

[0214] After the diabetic nephropathy model was successfully prepared, the rats were randomly divided into a disease group injected with normal saline (referred to as DKD), a treatment group injected with MSCs T (referred to as KDK+MSC T ), and a treatment group injected with MSCs EN (referred to as KDK+MSC T ), with unmodeled normal rats serving as a control group (Sham), with at least 5 rats in each group. The treatment groups were injected with 2 x 106 MSCs via the tail vein at weeks 11 and 12, and the control group was injected with 500 μL of normal saline.

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

[0216] The rats were weighed every week. The tail vein was punctured to measure the blood glucose. One day before the end of the experiment, the rats were put in metabolic cages to collect urine and feces, which were stored in a refrigerator at -80°C. After two weeks of continuous treatment, the rats were first weighed, then anesthetized, and the thoracic cavity was opened. The heart was punctured at the left auricle with a 5 mL syringe and the blood was collected into an EP tube. The tube was centrifuged at 300 x g for 10 min, and the supernatant was collected. After standing for 30 min, the supernatant was collected as serum and stored in a refrigerator at -80°C for later use. Then the abdominal cavity of the rat was opened and the kidneys and spleen were removed for weighing. Part of the kidney was immediately placed in liquid nitrogen for storage; part of the kidney was fixed in formaldehyde overnight, and the specimen was treated with 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, and anhydrous ethanol in order of low concentration to high concentration for 2 h. The tissue was dehydrated twice in anhydrous ethanol for 1 h each time, then placed in xylene for transparency, and the transparent tissue was placed in melted paraffin. After the paraffin completely immersed the tissue block, embedding was performed, and the cooled and fixed block was fixed on a microtome, sliced, and placed in heated water to flatten. The flattened tissue was attached to a glass slide and dried in a constant temperature oven at 45°C. The glass slide was stored at room temperature; part of the kidney was fixed in formaldehyde overnight, and the kidney was dehydrated in 30% sucrose solution until it sank to the bottom. The tissue was removed, washed twice with pre-cooled PBS, and immersed in OCT embedding agent. The pre-cooled OCT was fixed on a microtome, sliced, and attached to a glass slide for storage in a refrigerator at -80°C.

[0217] 1.4 Detection of biochemical indicators of rats

[0218] ① The kidney index was calculated according to the following formula:

[0219]

[0220] ② The spleen index was calculated according to the following formula:

[0221]

[0222] ③ Urine indicators:

[0223] After the rat urine was balanced to room temperature, the supernatant was obtained by centrifugation at 1000 x g for 10 min. The protein content in the rat urine was detected.

[0224] ④ Kidney indicators:

[0225] Part of the kidney tissue was added to 0.9% physiological saline, and the tissue was homogenized on ice with a tissue homogenizer at low speed to prepare a 10% tissue homogenate. The homogenate was centrifuged at 8000 x g and 4°C for 10 min, and the supernatant was collected for later use.

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

[0227] For superoxide dismutase (SOD) detection, the required reagents were added to a 96-well plate according to Table 3.4; after mixing well, it was incubated in a 37°C water bath for 30 min, and the absorbance of the sample at 450 nm was measured.

[0228] Table 3.4 Kidney Pathology Score Table

[0229]

[0230] 1.5 Rat Kidney Pathology Staining

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

[0232] 1.6 Rat Kidney Immunofluorescence Staining

[0233] After the prepared frozen sections were washed with running water for 5 min to remove OCT, immunofluorescence staining was performed according to the method of Example Three to detect the expression and distribution of synaptopodin in different kidney tissues.

[0234] 1.7 Rat Kidney Immunohistochemical Staining

[0235] The prepared frozen section was washed with running water for 5 min to wash away OCT; the section was placed in a wet box, 100 μL of 3% hydrogen peroxide was added dropwise, and incubated at room temperature for 10 min; washed with PBS for 3 times, 2 min each time, and distilled water for 3 min; spin-dried, 100 μL of goat serum was added for room temperature blocking for 10 min; the blocking solution was spun off, 100 μL of rabbit anti-NRF2 (1:1000 dilution) antibody was added dropwise, and incubated at 4°C overnight; washed with PBS for 3 times, 3 min each time, and distilled water for 3 min; spin-dried, 100 μL of HRP-labeled goat anti-rabbit IgG (H+L) antibody (1:1000 dilution) was added dropwise, and incubated at room temperature for 2 hrs; washed with PBS for 3 times, 3 min each time, and distilled water for 3 min; spin-dried, 100 μL of streptomyces anti-biotin-peroxidase solution was added dropwise, and incubated at room temperature for 10 min; washed with PBS for 3 times, 3 min each time, and distilled water for 3 min; 100 μL of DAB solution was added dropwise, and observed under a microscope for 3-10 min until the staining was appropriate; after washing with tap water, hematoxylin staining was performed for 2 min, and then counterstained with tap water; 1% hydrochloric acid alcohol was used for differentiation for 3 sec, and then washed with tap water for 3 min; 70% alcohol was used for treatment for 1 sec, 80% alcohol was used for treatment for 1 sec, 85% alcohol was used for treatment for 1 sec, 90% alcohol was used for treatment for 1 sec, 95% alcohol was used for treatment for 30 sec, and anhydrous alcohol was used for treatment for 30 sec for dehydration; air-dried in a ventilated place, soaked in xylene for 5 min, and then air-dried in a ventilated kitchen, and then mounted with neutral resin, and stored at room temperature.

[0236] 1.8 Rat kidney protein immunoblotting

[0237] First, 50 mg of kidney tissue was weighed and placed in a 1.5 mL centrifuge tube, and 0.5 mL of protein lysis buffer was added; homogenized in an ice bath using a tissue homogenizer; then centrifuged at 4°C, 12000 rpm for 15 min, and the supernatant was taken to measure the protein concentration and Western Blotting experiment according to the method of 2.3.7.2, and the expression of NRF2, HO-1, BCL-2, BAX, Nephrin and WT1 in different treated kidneys was detected at the protein level.

[0238] 2. Results

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

[0240] We constructed a rat DKD model by high-sugar feeding and injection of STZ after one week of adaptive feeding, and evaluated the immunomodulatory ability and anti-apoptotic potential of MSC EN . At the end of the two-week transplantation period, compared with rats in the untreated group and the MSC T treated group, MSC ENThe rats in the treatment group showed significant improvement in related diabetic symptoms such as urine, serum, etc., and the pathological symptoms of kidney tissue were reduced, and the progression of diabetic nephropathy was significantly delayed.

[0241] 2.2 MSC EN Effect of transplantation on physiological and biochemical indicators of type 2 diabetic rats

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

[0243] 2.3 MSC EN Effect of transplantation on kidney morphology of type 2 diabetic rats

[0244] Compared with DKD and MSC T treatment groups, the kidney weight and kidney weight index of rats in the MSC EN treatment group were significantly reduced ( Figure 30 ). H&E and PAS staining results showed that compared with the Sham group, the glomeruli of DKD rats were hypertrophic and the mesangial area matrix was significantly proliferated. Compared with the MSC T treatment group, the glomerular volume of rats in the MSC EN treatment group was basically normal and the mesangial matrix was significantly reduced. Masson staining results showed that compared with the Sham group, the glomerular mesangial area of DKD rats had increased collagen deposition, while the transplantation of MSC EN effectively reduced glomerular fibrosis ( Figure 31 ). These results indicate that MSC EN effectively improved the kidney damage of DKD rats and had a stronger protective effect on the kidneys of DKD rats.

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

[0246] Oxidative damage caused by hyperglycemia 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 increase in oxidative stress, lipid peroxidation is aggravated, and the activity of SOD is often affected. Compared with MSC T -treated rats, the content of MDA in the kidney of MSCEN-treated rats was significantly decreased, and the activity of SOD was increased Figure 32 , effectively reducing cell damage caused by oxidative stress and alleviating the development of diabetic nephropathy.

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

[0248] As shown in Figure 33 , the activity of NRF2 is often inhibited in DKD rats, leading to an increase in 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 the increase in oxidative stress. The transplantation of hMSCs can effectively restore the expression of NRF2 and further promote the expression of HO-1, reduce the BAX / Bcl-2 ratio, and reduce apoptosis in the kidney, thereby alleviating kidney function damage Figure 34 . Compared with MSC T -treated rats, the BAX / Bcl-2 ratio in the MSC EN -treated group was lower, demonstrating that hENFc enabled hMSCs with stronger anti-apoptotic ability.

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

[0250] Under hyperglycemic conditions, inflammatory reactions in the tissues of diabetic patients are activated, leading to increased inflammatory cell infiltration and release of inflammatory factors, which further aggravate the damage to kidney tissue and accelerate the development of diabetic nephropathy. To study whether the transplantation of hMSCs regulates the inflammatory response in DKD rats, we observed the spleen image Figure 35 B) and its weight Figure 35 A) and spleen index Figure 35 C) of the rats, and the results showed that the organ size and organ index of the spleen of MSC EN -treated mice were significantly lower than those of the other three groups. These results indicate that the transplantation of MSC EN has an inhibitory effect on splenomegaly, which may be related to the anti-inflammatory effect of MSC ENInhibition of lymphocyte proliferation was closely associated. We continued to determine the expression of pro-inflammatory cytokines in the kidney of rats, the results showed that the mRNA levels of IL-1β and TNF-α in the kidney of rats in the hMSCs treatment group were reduced compared with the DKD group. Cell damage and inflammatory factor release caused by inflammation can induce the activation of fibroblasts, leading to fibrosis of the renal interstitium. Our results showed that the expression of IL-1β and TNF-α in the kidney of rats in the MSCEN treatment group was reduced compared with the MSCT treatment group, which further reduced the expression of TGF-β Figure 35 D). Meanwhile, the concentrations of IL-1β, TNF-α and TGF-β in the serum of rats in the MSC EN treatment group were also observed to be reduced Figure 35 E). These results showed that MSC EN transplantation can effectively reduce the inflammatory response in the whole body and kidney tissue of DKD rats, thereby delaying the progression of diabetic nephropathy.

[0251] 2.7 Effect of MSC EN transplantation on kidney function in type 2 diabetic rats

[0252] Synaptopodin is related to maintaining the structure and function of the glomerular filtration membrane, and it is involved in regulating the morphology and dynamic structure of podocytes, which is essential for maintaining the filtration function of the glomerulus. Nephrin is closely related to the selective permeability of the glomerular filtration membrane, and it builds the interstitial membrane of the podocyte by interacting with other proteins, maintains the structural integrity of the filtration membrane, and is involved in regulating blood filtration and urine formation in the glomerulus. In the glomerulus, the expression of Wilms tumor gene 1 (WT1) is related to the formation of glomerular mother cells 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 tubular and interstitial cells. We found that, compared with the treatment of MSCT, the treatment of MSC EN restored the expression and distribution of Synaptopodin in the kidney of rats Figure 36 A), and increased the expression of Nephrin and WT1 proteins Figure 36 B). It was proved that MSC EN transplantation played an important role in repairing the filtration function of the glomerulus, maintaining the integrity of the filtration membrane, and regulating the structure and function of the glomerulus in DKD rats.

[0253] Example Six, MSC EN treatment of systemic sclerosis

[0254] 1. Experimental method

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

[0256] Select 6-8 weeks old, SFP level female mice, randomly divide C57BL mice 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 group). The normal control group of mice was injected with 100 μL of normal saline daily, and the disease control group and cell treatment group were injected with 100 μL of 1 mg / mL bleomycin daily, for 28 consecutive days.

[0257] Cells were treated by tail vein injection at a dose of 100,000 cells per mouse, starting from the success of modeling, once a week for a total of 2 times, and the treatment effect was detected 28 days after modeling.

[0258] 1.2 Cell co-culture model

[0259] Co-culture with mouse fibroblasts: Mouse fibroblasts were seeded in the lower chamber of a transwell, and when the cells grew to 70-80% confluence, 10 ng / mL TGF-β was used to construct the fibrosis group, and the cells were seeded in the upper chamber. The reverse fibrosis function was studied by PCR and WB methods. The PCR method refers to the method of Example II, and the following primers are used for 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 were seeded in a 48-well plate at a ratio of 1:10 with PBMC. After 3 days, the PBMC proliferation inhibition level was detected.

[0262] 2. Results

[0263] Under three-dimensional conditions, MSC EN cells can inhibit the proliferation of activated T cells, and MSC EN cells can increase the inhibition efficiency by about 30% compared with the Control group, as shown in Figure 37 . MSC EN reverses the fibrosis phenotype of skin fibroblasts, inhibits fibrosis-related gene expression, and down-regulates Col1A1 by about 25% and α-SMA by about 17%, as shown in Figure 38 . Compared with the MSC T group, MSC EN cells slow down the immune infiltration at the lesion site (reduce 6.5% of pro-inflammatory related CD4+ lymphocytes, inhibit 0.17% of Th1 lymphocytes, and enhance 0.2% of anti-inflammatory type regulatory T lymphocytes Treg cells), as shown in Figure 39 . MSCEN Cell reversed the degree of fibrosis of skin fibroblasts (about 20% reduction in dermal layer thickness, about 1.5-fold increase in subcutaneous fat layer thickness), such as Figure 40 MSC EN Cell effectively reduced collagen deposition in lung tissue, reversed the degree of fibrosis of the tissue (about 30% down-regulation of COL1A1 and COL1A2, about 25% down-regulation of COL3A1, and about 20% down-regulation of α-SMA), such as Figure 41 MSC EN Cell significantly improved the integrity and permeability of blood vessels, such as Figure 42 .

[0264] Example Seven, MSC EN Treatment of Sjogren's syndrome

[0265] 1. Sjogren's syndrome mouse modeling and treatment:

[0266] ① Select 8-week-old, SFP grade female NOD mice, feed in a constant temperature condition without special pathogen, and determine whether they are sick by measuring the saliva flow rate.

[0267] ② After confirming the onset, MSCs T and MSC EN are treated by tail vein injection at a dose of 100,000 cells per mouse once, and the material is taken 21 days after modeling.

[0268] 2. Results

[0269] MSC EN Cell improves the function of salivary glands in Sjogren's syndrome, up-regulates the flow rate of salivary glands to increase the salivary gland / body weight ratio, reduces salivary gland damage and collagen deposition, reduces inflammatory cell infiltration, reduces acinar and duct atrophy, reduces the degree of fibrosis, increases aquaporin expression, and relieves salivary gland function, such as Figure 43 .

[0270] In summary, the present application provides a use of a matrix-enabled mesenchymal stem cell in the preparation of a drug for preventing and treating immune diseases, wherein the mesenchymal stem cell is obtained by culturing and enabling the cell culture matrix hENFc, 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 cell can promote the nuclear translocation of β-catenin, enhance the proliferation ability, stem maintenance ability, delay cell aging and apoptosis of in vitro expansion, hMSCs cultured to the eleventh generation by hENFc enablement do not have tumorigenicity and do not have any adverse effects on animals, and meet the clinical application standards of hMSCs. The matrix-enabled mesenchymal stem cell not only has better survival ability in the damaged microenvironment, but also has stronger environmental perception and anti-inflammatory ability, and significantly inhibits PBMC proliferationFigure 5 A) and promoting apoptosis Figure 5 B), MSC EN The proportion of CD4+ and CD8+ T cell subsets is also inhibited, improving the efficacy of hMSCs in inflammatory and autoimmune diseases. The stromal-enabled mesenchymal stem cells can treat systemic lupus erythematosus nephritis, and after treatment, the glomerular mesangial and basement membrane thickening, inflammatory cell infiltration, interstitial fibrosis, and occasional crescent formation of the mouse are significantly reduced; the stromal-enabled mesenchymal stem cells can treat diabetic nephropathy, and after treatment, the histopathological symptoms of the mouse kidney are reduced, and the progression of diabetic nephropathy is significantly delayed; the increase of blood glucose, 24-hour urinary protein, BUN and SCr of rats is significantly inhibited; the potential of hMSCs to slow down kidney damage in diabetic rats is effectively improved, the kidney function of DKD rats is improved, the cell damage caused by oxidative stress is effectively reduced, the development of diabetic nephropathy is alleviated, and the anti-apoptotic ability is stronger. The stromal-enabled mesenchymal stem cells can inhibit the proliferation of activated T cells, MSC EN cells compared to MSC T The inhibition efficiency is increased by about 30%, the fibrosis phenotype of skin fibroblasts is reversed, fibrosis-related gene expression is inhibited, Col1A1 is down-regulated by about 25%, and a-SMA is down-regulated by about 17%, the immune infiltration at the lesion site is slowed down (pro-inflammatory related CD4+ lymphocytes are reduced by 6.5%, Th1 lymphocytes are inhibited by 0.17%, and anti-inflammatory regulatory T lymphocytes Treg cells are enhanced by 0.2%), the degree of fibrosis of skin fibroblasts is reversed (the thickness of the dermal layer is reduced by about 20%, and the thickness of the subcutaneous fat layer is increased by about 1.5 times); collagen deposition in skin and lung tissue is effectively reduced, and the degree of fibrosis of the tissue is reversed (COL1A1 and COL1A2 are down-regulated by about 30%, COL3A1 is down-regulated by about 25%, and a-SMA is down-regulated by about 20%); the integrity and permeability of blood vessels are significantly improved. The stromal-enabled mesenchymal stem cells can improve the function of the salivary glands of xerostomia, up-regulate the flow rate of the salivary glands, reduce inflammatory cell infiltration, reduce atrophy of acinar and ductal glands, reduce the degree of fibrosis, and increase the expression of aquaporin, and the function of the salivary glands is alleviated.

Claims

1. The use of matrix-primed mesenchymal stem cells in the preparation of a drug for preventing and treating autoimmune diseases, characterized in that, The mesenchymal stem cells are obtained by culturing mesenchymal stem cells in a cell culture medium hENFc, wherein the cell culture medium hENFc is prepared from 1-3 parts of hE-cad-Fc and 1-3 parts of hN-cad-Fc, wherein the cell culture medium hENFc is diluted to a total protein concentration of 1-30 μg / mL, and then added to a tissue culture treated plate and incubated for 30-150 min, and then washed to obtain the cell culture medium hENFc, and the autoimmune disease is one or more of systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome and diabetic nephropathy.

2. Use according to claim 1, wherein The mesenchymal stem cells are derived from a mammal.

3. Use according to claim 2, wherein the compound is ###0002### The mesenchymal stem cells are derived from a human, a pig or a mouse.

4. Use according to claim 3, wherein the compound is ###0002### The mesenchymal stem cells are derived from bone marrow, adipose tissue, dental pulp, umbilical cord, umbilical cord blood, amniotic fluid or placenta.

5. The use according to claim 4, wherein the compound is ###0002### The mesenchymal stem cells are derived from umbilical cord or umbilical cord blood. The mesenchymal stem cells are derived from umbilical cord or umbilical cord blood.

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