CN109593723B

CN109593723B - Mesenchymal stem cell for inhibiting immune reaction and preparation method and application thereof

Info

Publication number: CN109593723B
Application number: CN201811547307.8A
Authority: CN
Inventors: 江小霞; 王常勇; 周瑾; 王玉涵; 黄小会; 王�华; 党瑞杰; 何有娣
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2018-12-18
Filing date: 2018-12-18
Publication date: 2022-03-01
Anticipated expiration: 2038-12-18
Also published as: CN109593723A

Abstract

The invention discloses mesenchymal stem cells for efficiently inhibiting immune response and a preparation method and application thereof. The preparation method of the recombinant mesenchymal stem cell comprises the following steps: reducing the expression quantity of microRNA-129 genes in the receptor mesenchymal stem cells to obtain recombinant mesenchymal stem cells; the receptor mesenchymal stem cell is an in vitro mesenchymal stem cell. The preparation method is simple to operate, convenient and practical, the obtained mesenchymal stem cells are immunosuppressive mesenchymal stem cells, the secretion capacity of immune factors is reduced, and the NO secretion capacity is obviously enhanced, so that the capacity of inhibiting inflammatory reactions (such as inflammatory enteritis) is enhanced. Therefore, the invention establishes a stable preparation method of the mesenchymal stem cells for efficiently inhibiting the immune response, and lays a foundation for the research and the application of the mesenchymal stem cells.

Description

Mesenchymal stem cell for inhibiting immune reaction and preparation method and application thereof

Technical Field

The invention relates to mesenchymal stem cells for efficiently inhibiting immune response and a preparation method and application thereof.

Background

Mesenchymal Stem Cells (MSCs) are adult stem cells with a strong self-renewal and multipotential differentiation capacity. A large number of researches find that the MSCs also have an immune regulation function, the MSCs have a strong inhibition effect on an immune system after being stimulated by inflammatory cytokines, and the MSCs have good treatment effects on a plurality of inflammatory-related and immune-related diseases such as graft-versus-host disease (GvHD), inflammatory enteritis (IBD), Type I Diabetes and the like. Therefore, MSC can be used for reducing immunological rejection, prolonging survival time of transplant, and treating related immune disorder, such as autoimmune disease.

MSCs are capable of secreting large amounts of chemokines and Nitric Oxide (NO) with immunosuppressive properties under stimulation by inflammatory factors. Under the action of chemokines, immune cells are chemotactic to the periphery of MSCs and inhibited by their local high concentration of NO. In recent studies, however, MSCs have been found to promote immune responses under certain conditions, and if sufficient pro-inflammatory cytokines are not present in the MSC-based environment to induce sufficient NO production, MSCs are able to enhance immune responses, which has been well documented in vitro cell proliferation assays and in animal models of delayed hypersensitivity diseases.

mirnas (micrornas ) are a class of endogenous single-stranded non-coding small RNA of about 20-24nt in length, which are widely found in organisms. In recent years, a great deal of research shows that miRNA can regulate the expression of a plurality of genes in organisms, and plays an important role in regulating growth and development, cell proliferation, apoptosis, resisting environmental stress and the like.

The microRNA-129 (microRNA-129, miR-129) family consists of two members: miR-129-1(Gene ID:406917) located on chromosome 7q32.1 and miR-129-2(Gene ID: 406918) located on chromosome 11p11.2, which contain a common seed sequence "UUUUGC". miR-129-2 is located on the CpG island at the 5' end, while 7q32.1 is usually deleted in tumors. MiR-129 family members are generally considered tumor suppressors and lack expression in a variety of tumor tissues. miR-129-5p is the main product and functional form of miR-129. To date, the function research of miR-129 mainly focuses on the regulation and control effect of the miR-129 on the occurrence and development of tumors.

Disclosure of Invention

The technical problem to be solved by the invention is how to enhance the capability of mesenchymal stem cells for inhibiting immune response (such as inhibiting inflammatory response).

In order to solve the technical problems, the invention provides a preparation method of a recombinant mesenchymal stem cell.

The preparation method of the recombinant mesenchymal stem cells provided by the invention comprises the following steps: reducing the expression quantity of microRNA-129 genes in the receptor mesenchymal stem cells to obtain recombinant mesenchymal stem cells; the receptor mesenchymal stem cell is an in vitro mesenchymal stem cell.

In the preparation method, the nucleotide sequence of the microRNA-129 can be SEQ ID No. 1.

In the preparation method, the step of reducing the expression level of the microRNA-129 gene in the receptor mesenchymal stem cell comprises the step of introducing a substance for inhibiting the expression of the microRNA-129 gene into the receptor mesenchymal stem cell.

In the preparation method, the recombinant mesenchymal stem cells can have at least one of the following characteristics A1) -A5):

A1) the nitric oxide secretion capacity of the recombinant mesenchymal stem cell is higher than that of the receptor mesenchymal stem cell;

A2) the recombinant mesenchymal stem cell has higher immunosuppressive capacity than the receptor mesenchymal stem cell;

A3) the recombinant mesenchymal stem cell has a lower expression capacity of proinflammatory factors (proinflammatory cytokines) than the receptor mesenchymal stem cell;

A4) the recombinant mesenchymal stem cell has higher anti-inflammatory factor (anti-inflammatory factor or anti-inflammatory cytokine) expression capacity than the receptor mesenchymal stem cell;

A5) the recombinant mesenchymal stem cell has higher inflammatory injury weakening capability than the receptor mesenchymal stem cell.

In the preparation method, in the treatment of 0ng/mL of TNF-alpha and 0ng/mL of IFN-gamma, the nitric oxide secretion capacity of the recombinant mesenchymal stem cell can be 1.6-2.5 times that of the receptor mesenchymal stem cell; in the treatment of 5ng/mL TNF-alpha and 5ng/mL IFN-gamma, the nitric oxide secreting ability of the recombinant mesenchymal stem cell may be 2.0-3.3 times that of the recipient mesenchymal stem cell; in the treatment of 10ng/mL TNF-alpha and 10ng/mL IFN-gamma, the nitric oxide secreting ability of the recombinant mesenchymal stem cell may be 1.8-2.0 times that of the recipient mesenchymal stem cell; the nitric oxide secretion capacity of the recombinant mesenchymal stem cells was 1.9 times that of the recipient mesenchymal stem cells in the treatment of 0 μ g/mL of LPS, in the treatment of 1.0 μ g/mL of LPS and in the treatment of 1.5 μ g/mL of LPS.

In the above preparation method, the proinflammatory factor can be TNF-alpha and/or IFN-gamma, and the TNF-alpha expression capacity of the recombinant mesenchymal stem cell can be 1/10 of the receptor mesenchymal stem cell in the treatment of 0ng/mL of TNF-alpha and 0ng/mL of IFN-gamma, the treatment of 5ng/mL of TNF-alpha and 5ng/mL of IFN-gamma and the treatment of 10ng/mL of TNF-alpha and 10ng/mL of IFN-gamma; the recombinant mesenchymal stem cell may have a TNF-a expression capacity 1/16-fold that of the recipient mesenchymal stem cell in the treatment with 1.5 μ g/mL LPS; in the treatment of LPS of 1.5 μ g/mL, the recombinant mesenchymal stem cell may have 1/14-fold greater IFN- γ expression capacity than the recipient mesenchymal stem cell.

The inflammation-inhibiting factor is IL-10 and/or iNOS; in the treatment of 5ng/mL of TNF- α and 5ng/mL of IFN- γ, the IL-10 expression capacity of the recombinant mesenchymal stem cell may be 10 times that of the recipient mesenchymal stem cell, and the iNOS expression capacity of the recombinant mesenchymal stem cell may be 30 times that of the recipient mesenchymal stem cell; in the treatment of TNF- α of 10ng/mL and IFN- γ of 10ng/mL, the IL-10 expression capacity of the recombinant mesenchymal stem cell may be 8 times that of the receptor mesenchymal stem cell, and the iNOS expression capacity of the recombinant mesenchymal stem cell may be 16 times that of the receptor mesenchymal stem cell.

In the above preparation method, the inflammatory injury may be inflammatory enteritis.

In the preparation method, the substance for inhibiting the expression of the microRNA-129 gene can be any one of the following biological materials B1) -B5):

B1) RNA of which the nucleotide sequence is SEQ ID No. 3;

B2) an expression vector expressing the RNA of B1);

B3) a recombinant microbial cell expressing the RNA of B1);

B4) recombinant animal cells expressing the RNA of B1);

B5) recombinant plant cells expressing the RNA of B1).

In the preparation method, the substance for inhibiting the expression of the MicroRNA-129 gene in the receptor mesenchymal stem cell can be a recombinant lentivirus for expressing the RNA.

In the above preparation method, the isolated mesenchymal stem cell may be an isolated mesenchymal stem cell of a mammal.

The recombinant mesenchymal stem cells prepared by the preparation method also belong to the protection scope of the invention.

Any of the following applications also fall within the scope of the present invention:

p1, the application of the recombinant mesenchymal stem cell in the preparation of immunosuppressive products (medicines or vaccines);

p2, use of the recombinant mesenchymal stem cells in the preparation of a product (medicament or vaccine) for attenuating inflammatory lesions;

p3, and the application of the substance for inhibiting the microRNA-129 gene expression in the preparation of products (drugs or vaccines) for enhancing the nitric oxide secretion capacity of mesenchymal stem cells;

p4, and the application of the substance for inhibiting the expression of the microRNA-129 gene in the preparation of products (drugs or vaccines) for enhancing the immunosuppressive capability of mesenchymal stem cells;

p5, and the application of the substance for inhibiting the expression of the microRNA-129 gene in the preparation of products (medicines or vaccines) for reducing the expression capacity of proinflammatory factors of mesenchymal stem cells;

p6, and the application of the substance for inhibiting the expression of the microRNA-129 gene in the preparation of products (drugs or vaccines) for enhancing the anti-inflammatory factor expression ability of mesenchymal stem cells.

In the above P3, the capacity of enhancing the secretion of nitric oxide of the mesenchymal stem cell may be 0.6 to 2.3 times of the capacity of enhancing the secretion of nitric oxide of the mesenchymal stem cell; specifically, under the conditions of 0ng/mL of TNF-alpha and 0ng/mL of IFN-gamma, the nitric oxide secretion capacity of the mesenchymal stem cells is enhanced by 0.6-1.5 times; under the conditions of 5ng/mL of TNF-alpha and 5ng/mL, the nitric oxide secretion capacity of the mesenchymal stem cells is enhanced by 1.0-2.3 times; enhancing the nitric oxide secretion capacity of the mesenchymal stem cells by 0.8-1.0 times under the conditions of 10ng/mL of TNF-alpha and 10ng/mL of IFN-gamma; enhancing the nitric oxide secretion capacity of the mesenchymal stem cells by 0.9 times under the condition of 0 mu g/mL of LPS, 1.0 mu g/mL of LPS and 1.5 mu g/mL of LPS.

In the above P5, the proinflammatory factor can be TNF-alpha and/or IFN-gamma; the reduction of the proinflammatory factor expression capacity of the mesenchymal stem cell can be 0.9 times of the reduction of the TNF-alpha expression capacity of the mesenchymal stem cell and/or 0.9 times of the reduction of the IFN-gamma expression capacity of the mesenchymal stem cell, and specifically, the reduction of the TNF-alpha expression capacity of the mesenchymal stem cell is 0.9 times of the reduction of the TNF-alpha expression capacity of the mesenchymal stem cell under the conditions of 0ng/mL of TNF-alpha and 0ng/mL of IFN-gamma, 5ng/mL of TNF-alpha and 5ng/mL of IFN-gamma, and 10ng/mL of TNF-alpha and 10ng/mL of IFN-gamma; reducing the TNF-alpha expression capacity of the mesenchymal stem cells by 0.9 times under the condition of 1.5 mu g/mL of LPS; reducing the TNF-alpha expression capacity of the mesenchymal stem cells by 0.9 times under the condition of 1.5 mu g/mL of LPS; the IFN-gamma expression capacity of the mesenchymal stem cells is reduced by 0.9 times under the condition of 1.5 mu g/mL of LPS.

In the above P6, the inflammation inhibitor may be IL-10 and/or iNOS; the anti-inflammatory factor expression capacity of the enhanced mesenchymal stem cell can be 7-9 times of the IL-10 expression capacity of the enhanced mesenchymal stem cell and/or 15-29 times of the iNOS expression capacity of the enhanced mesenchymal stem cell, specifically, 9 times of the IL-10 expression capacity of the enhanced mesenchymal stem cell and 29 times of the iNOS expression capacity of the enhanced mesenchymal stem cell under the conditions of 5ng/mL of TNF-alpha and 5ng/mL of IFN-gamma; under the conditions of 10ng/mL of TNF-alpha and 10ng/mL of IFN-gamma, the IL-10 expression capacity of the mesenchymal stem cells is enhanced by 7 times, and the iNOS expression capacity of the mesenchymal stem cells is enhanced by 15 times.

In the above application, the inflammatory injury may be inflammatory enteritis.

As above, the immunosuppression may be delayed-type hypersensitivity inhibition.

According to the invention, the reactivity of the mesenchymal stem cells to inflammatory factors is enhanced by interfering the expression of microRNA-129(miR-129) in the mesenchymal stem cells, the NO secretion capacity of the mesenchymal stem cells is greatly improved, and the inhibition of inflammatory reaction (inflammatory enteritis) is enhanced, so that the capacity of inhibiting immune reaction is enhanced. The recombinant mesenchymal stem cells with high-efficiency immune reaction inhibition obtained by the invention can be stably passed, frozen and restored. The preparation method is simple to operate, convenient and practical, the obtained mesenchymal stem cells are immunosuppressive mesenchymal stem cells, the secretion capacity of immune factors is reduced, and the NO secretion capacity is obviously enhanced, so that the capacity of inhibiting inflammatory reaction (inflammatory enteritis) is enhanced. The invention establishes a stable preparation method of the mesenchymal stem cells for efficiently inhibiting the immune response, and lays a foundation for the research and the application of the mesenchymal stem cells.

Drawings

FIG. 1 shows that IBD model detects the difference of the in vivo immunoregulatory capacities of MSC-LV-GFP group and MSC-LV-ShmmiR-129-the rate of change of body weight.

FIG. 2 shows that IBD model detects the difference in immunomodulatory capacity between the MSC-LV-GFP group and MSC-LV-ShmmiR-129 in vivo-pathological index.

FIG. 3 shows that IBD model detects differences in the immunomodulatory capacity of the MSC-LV-GFP group and MSC-LV-ShmmiR-129 in vivo-colon length, colon weight and spleen weight.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The mouse MSC cell line C3H/10T1/2 cells in the following examples are products of cell resource center of Shanghai Life sciences research institute of Chinese academy of sciences, and the catalog number is SCSP-506.

α -MEM culture medium (Gibco, cat. No.12000-022) in the following examples; both TNF-alpha and INF-gamma are products of Perotech corporation; propylene glycol methyl ether acetate (PMA) and ionomycin are both products of eBioscience; CFSE is Invitrogen; .

The quantitative data in the following examples were analyzed using SPSS13.0 software, the mean of the samples was compared using one-way anova, the mean of two samples between groups was compared using SNK, and P <0.05 was statistically significant for differences. P <0.05, P < 0.01.

The following examples illustrate the technical scheme of the present invention by taking mouse-derived mmu-miR-129-5p (also referred to as mmu-miR-129) as an example.

The microRNA-129 in the following examples is mmu-miR-129-5p, whose Access number at miRBase is MIMAT0000209, and whose nucleotide sequence is 5'-cuuuuugcggucugggcuugc-3' (SEQ ID No. 1). The microRNA-129 gene is 5'-ctttttgcggtctgggcttgc-3'.

Example 1 reduction of expression level of microRNA-129 Gene in receptor mesenchymal Stem cell to obtain recombinant mesenchymal Stem cell

In this example, fat-derived C57BL/6 mouse primary mesenchymal stem cells (hereinafter abbreviated as MSC) and mouse MSC cell line C3H/10T1/2 cells (hereinafter abbreviated as C3H10) were used as recipient mesenchymal stem cells, and recombinant lentiviruses expressing RNA of the MicroRNA-129 gene were introduced into the recipient mesenchymal stem cells, thereby obtaining recombinant mesenchymal stem cells in which the expression level of the MicroRNA-129 gene was reduced as compared with the recipient mesenchymal stem cells.

The specific method comprises the following steps:

1. preparation of recombinant mesenchymal stem cells by MicroRNA-129 gene silencing

1.1 preparation of recombinant lentivirus LV-ShmmiR-129

Artificially synthesizing a DNA fragment mmu-miR-129-5p-inhibition for inhibiting MicroRNA-129 gene expression:

5’-AATTCAAAAACTTTTTGCGGTCTGGGCTTGC-3’

3’-gTTTTTGAAAAACGCCAGACCCGAACGggcC-5’。

the mmu-miR-129-5p-inhibition is inserted between Age I and EcoR I recognition sites of a lentiviral vector GV280 (Shanghai Jikai Gene science and technology Co., Ltd.) to obtain a recombinant vector, and the recombinant vector is named as GV280-miR-129 down. GV280-mir-129down contains DNA having the following nucleotide sequence:

5'-GCAAGCCCAGACCGCAAAAAG-3' (SEQ ID No.2), expressing an RNA having the nucleotide sequence:

5’-GCAAGCCCAGACCGCAAAAAG-3’(SEQ ID No.3)

GV280-mir-129down, pHelper1.0 vector and pHelper2.0 vector (Shanghai Jikai gene technology Co., Ltd.) were co-transfected into 293T cells to obtain recombinant lentivirus LV-ShmmiR-129 (capable of interfering with the expression of MicroRNA-129 gene). And determining the virus titer according to the expression quantity of the green fluorescent protein of the 293T cell.

1.2 preparation of recombinant lentivirus LV-GFP

Artificially synthesizing a DNA fragment CON137 for inhibiting the expression of a control gene:

5’-AATTCAAAAAACGTGACACGTTCGGAGAA-3’

3’-gTTTTTTGCACTGTGCAAGCCTCTTggcC-5’。

CON137 was inserted between the Age I and EcoR I recognition sites of lentiviral vector GV280 (Shanghai Kjeka Gene science, Inc.) to obtain a recombinant vector, which was named GV 280-mir-CONdown.

GV280-mir-CONdown, pHelper1.0 vector and pHelper2.0 vector (Kyoto Gene technology Co., Ltd., Shanghai) were co-transfected into 293T cells to obtain recombinant lentivirus LV-GFP (control of LV-ShmmiR-129). And determining the virus titer according to the expression quantity of the green fluorescent protein of the 293T cell.

1.3 preparation of recombinant mesenchymal Stem cell MSC-LV-ShmmiR-129

MSC was suspended in α -MEM containing 10% (by volume) fetal bovine serum to obtain MSC cell suspension. MSC cell suspension was adjusted to 2X 10⁵Per cm²The density of (A) was inoculated in a 6-well plate and incubated at 37 ℃ with 5% CO₂Culturing in a constant temperature incubator, adding LV-ShmmiR-129 recombinant lentivirus solution (MOI is 5) and polybrene (working concentration is 8 mug/mL) when the cells reach 80% fusion, continuously culturing for 6 hours, removing the original culture solution by suction, adding an equal volume of alpha-MEM culture solution containing 10% (volume percentage) fetal calf serum, placing at 37 ℃ and 5% CO₂The recombinant mesenchymal stem cells with the MicroRNA-129 gene silencing are obtained by subculture in a constant temperature incubator and are named as MSC-LV-ShmmiR-129.

1.4 preparation of control recombinant mesenchymal Stem cell MSC-LV-GFP

Replacing the LV-ShmmiR-129 recombinant lentivirus liquid of 1.3 with LV-GFP recombinant lentivirus liquid, and performing other operations in the same way as 1.3 to obtain recombinant mesenchymal stem cells, namely MSC-LV-GFP which is used as the control of the MSC-LV-ShmmiR-129.

1.5 preparation of recombinant mesenchymal Stem cell C3H10-LV-ShmmiR-129

The MSC of 1.3 is replaced by C3H10, and other operations are the same as 1.3, so that the recombinant mesenchymal stem cell is obtained and named as C3H 10-LV-ShmmiR-129.

1.6 preparation of control recombinant mesenchymal Stem cell MSC-LV-GFP

The MSC of 1.4 is replaced by C3H10, and other operations are the same as 1.4, so that the recombinant mesenchymal stem cell which is named as C3H10-LV-GFP is obtained as the control of C3H 10-LV-ShmmiR-129.

Identification of di-and recombinant mesenchymal stem cells

1. Identification of MicroRNA-129 gene silencing efficiency in recombinant mesenchymal stem cells

The MSC-LV-ShmmiR-129 cells cultured to the third generation are cracked by a Tripure reagent, cell lysate is collected, RNA is extracted and is reversely transcribed into cDNA, the mRNA expression quantity of the MicroRNA-129 gene in the cells is detected by Real-time fluorescent Quantitative PCR (QRT-PCR), the mRNA expression quantity is expressed relative to the mRNA expression quantity of the U6 gene, so that the silencing efficiency of the gene is detected, and the experiment is repeated three times.

Wherein, MicroRNA-129 gene primer:

forward: 5'-CTTTTTGCGGTCTGGGCTTGC-3', respectively; the downstream primer is a universal primer and is derived from a miRNA fluorescent quantitative PCR kit (dye method) of biological engineering, ltd, and the product number is as follows: B532461.

the method for identifying the MicroRNA-129 gene silencing efficiency of the MSC, the MSC-LV-GFP, the C3H10-LV-ShmmiR-129 and the C3H10 cells is the same as that of the MSC-LV-ShmmiR-129 cells.

The experimental results are as follows: the mRNA expression quantity of the MicroRNA-129 gene in MSC cells relative to the mRNA expression quantity of the U6 gene is 1.0, the mRNA expression quantity of the MicroRNA-129 gene in MSC-LV-GFP relative to the mRNA expression quantity of the U6 gene is 1.0, and the mRNA expression quantity of the MicroRNA-129 gene in MSC-LV-ShmmiR-129 cells relative to the mRNA expression quantity of the U6 gene is 0.31; the expression quantity of mRNA of the MicroRNA-129 gene in the MSC-LV-ShmmiR-129 cells is obviously lower than that of the MSC cells and the MSC-LV-GFP cells.

The expression amount of mRNA of MicroRNA-129 gene in C3H10 relative to that of U6 gene is 1.0, the expression amount of mRNA of MicroRNA-129 gene in C3H10-LV-GFP relative to that of U6 gene is 1.0, and the expression amount of mRNA of MicroRNA-129 gene in C3H10-LV-ShmmiR-129 cell relative to that of U6 gene is 0.37; the expression level of mRNA of MicroRNA-129 gene in C3H10-LV-ShmmiR-129 cells is obviously lower than that of C3H10 cells and C3H10-LV-GFP cells.

2. Immune-related factor level determination

2.1 fluorescent quantitative PCR detection of TNF-alpha, IL-10 and iNOS (nitric oxide synthase) Gene expression levels in MSC-LV-ShmmiR-129 cells, MSC-LV-GFP cells and MSC cells

Culture solution N1: alpha-MEM containing 10% (volume percentage content) fetal calf serum, 0ng/mL TNF-alpha and 0ng/mL IFN-gamma.

Culture solution N2: alpha-MEM containing 10% (volume percentage content) fetal bovine serum, 5ng/mL TNF-alpha and 5ng/mL IFN-gamma.

Culture solution N3: alpha-MEM containing 10% (volume percentage content) fetal bovine serum, 10ng/mL TNF-alpha and 10ng/mL IFN-gamma.

Culturing 3 cells of MSC-LV-ShmmiR-129 cells, MSC-LV-GFP cells and MSC cells infected by the recombinant virus obtained in the step 1 for 72 hours by using a culture solution N (the culture solution N is culture solution N1, culture solution N2 or culture solution N3) independently and culturing according to the ratio of 1.5 x 10⁵Per cm²The cells were seeded in 6-well plates at 37 ℃ in 5% CO₂After culturing for 12h in the constant temperature incubator, taking the same number of cells, and carrying out real-time fluorescence quantitative PCR reaction according to the following method to detect the expression levels of TNF-alpha, IL-10 and iNOS genes in the cells:

total RNA from cells was extracted with TRIZOL and reverse transcribed into cDNA using a reverse transcriptase kit. The cDNA was used as a template to add a TOYOBO brand SYBR Green reagent to determine the expression of a specific gene using real-time fluorescent quantitative PCR, expressed as the amount of expression relative to the β -actin gene. The experiment was repeated three times:

wherein, the mouse beta-actin gene primer:

Forward：5’-CTTCCGCCTTAATACTTC-3’，

Reverse：5’-AAGCCTTCATACATCAAG-3’。

TNF-alpha gene primer:

Forward：5’-GATGGGTTGTACCTTGTCTACT-3’；

Reverse：5’-CTTTCTCCTGGTATGAGATAGC-3’。

iNOS gene primers:

Forward：5’-CAGCTGGGCTGTACAAACCTT-3’；

Reverse：5’-CATTGGAAGTGAAGCGTTTCG-3’。

IL-10 gene primers:

Forward：5’-CCAAGCCTTATCGGAAATGA-3’；

Reverse：5’-TCTCACCCAGGGAATTCAAA-3’。

the results showed that the same number of cells, in the treatment of 0ng/mL of TNF-alpha and 0ng/mL of IFN-gamma, in the treatment of 5ng/mL of TNF-alpha and 5ng/mL of IFN-gamma and in the treatment of 10ng/mL of TNF-alpha and 10ng/mL of IFN-gamma, the expression level of the TNF-alpha gene of MSC-LV-ShmmiR-129 cells was 1/10 times that of MSC cells of the receptor mesenchymal stem cells, and there was no significant difference in the expression level of the TNF-alpha gene between the MSC-LV-GFP cells and the MSC cells.

The same number of cells, in 0ng/mL TNF-alpha and 0ng/mL IFN-gamma treatment, IL-10 gene expression in MSC-LV-ShmmiR-129 cells, MSC-LV-GFP cells and MSC cells between no significant difference; in the treatment of 5ng/mL of TNF-alpha and 5ng/mL of IFN-gamma, the expression quantity of the IL-10 gene of the MSC-LV-ShmmiR-129 cell is 10 times that of the MSC cell of the receptor mesenchymal stem cell, and the expression quantity of the IL-10 gene between the MSC-LV-GFP cell and the MSC cell has no significant difference; in the same number of cells, the IL-10 gene expression amount of MSC-LV-ShmmiR-129 cells was 8 times that of MSC cells of the recipient mesenchymal stem cells in the treatment of 10ng/mL of TNF-alpha and 10ng/mL of IFN-gamma, and there was no significant difference in the IL-10 gene expression amount between the MSC-LV-GFP cells and the MSC cells.

In the same number of cells, iNOS gene was not expressed in MSC-LV-ShmmiR-129 cells, MSC-LV-GFP cells and MSC cells in the treatment of 0ng/mL TNF-. alpha.and 0ng/mL IFN-. gamma.; the expression level of iNOS gene of MSC-LV-ShmmiR-129 cells in the treatment of 5ng/mL TNF-alpha and 5ng/mL IFN-gamma of the same number of cells is 30 times of that of MSC cells of receptor mesenchymal stem cells, and the expression level of iNOS gene between the MSC-LV-GFP cells and the MSC cells has no significant difference; in the same number of cells, the expression amount of iNOS gene of MSC-LV-ShmmiR-129 cells was 16 times that of MSC cells of recipient mesenchymal stem cells in the treatment of 10ng/mL of TNF-alpha and 10ng/mL of IFN-gamma, and there was no significant difference in the expression amount of iNOS gene between the MSC-LV-GFP cells and the MSC cells.

After miR-129 expression is interfered, the expression quantity of proinflammatory factor TNF-alpha gene of the recombinant mesenchymal stem cell MSC-LV-ShmmiR-129 cell is 1/10 of the receptor mesenchymal stem cell MSC cell under the stimulation of inflammatory factors by the same number of cells, and the expression capacity of the proinflammatory factor IL-10 and iNOS is obviously more than ten times higher than that of the receptor mesenchymal stem cell MSC.

2.2 detection of nitric oxide secretion Capacity of MSC-LV-ShmmiR-129 cells, MSC-LV-GFP cells and MSC cells

Step 1 is to obtainAfter 72 hours of infection with the recombinant virus, 3 kinds of cells, namely, MSC-LV-ShmmiR-129 cells, MSC-LV-GFP cells and MSC cells, were cultured in culture medium N alone (culture medium N is culture medium 1, culture medium 2 or culture medium 3) at 1.5X 10⁵Per cm²The cells were seeded in 6-well plates at 37 ℃ in 5% CO₂After 12h incubation in the incubator, the supernatant was taken and NO was determined using Griess reagent (Sigma-Aldrich). In short, all NO₃Conversion to NO by NO reductase₂And total NO₂Detection was by Griess reaction.

The results showed that the same number of cells, in the treatment of 0ng/mL of TNF-alpha and 0ng/mL of IFN-gamma, the content of NO in the supernatant of MSC-LV-ShmmiR-129 cells was 7.50. mu.M, the content of NO in the supernatant of MSC-LV-GFP cells was 3.25. mu.M, the content of NO in the supernatant of MSC cells was 3.00. mu.M, and the NO secretion capacity of MSC-LV-ShmmiR-129 was 2.5 times that of MSC cells of the recipient mesenchymal stem cells; in the treatment of 5ng/mL TNF-alpha and 5ng/mL IFN-gamma, the content of NO in the supernatant of the MSC-LV-ShmmiR-129 cell is 18.00 mu M, the content of NO in the supernatant of the MSC-LV-GFP cell is 5.60 mu M, the content of NO in the supernatant of the MSC cell is 5.50 mu M, and the NO secretion capacity of the MSC-LV-ShmmiR-129 is 3.3 times that of the MSC cell of the receptor mesenchymal stem cell; in the same number of cells, in the treatment of 10ng/mL of TNF-alpha and 10ng/mL of IFN-gamma, the content of NO in the supernatant of the MSC-LV-ShmmiR-129 cells was 22.45. mu.M, the content of NO in the supernatant of the MSC-LV-GFP cells was 12.50. mu.M, the content of NO in the supernatant of the MSC-cells was 12.48. mu.M, and the NO secretion capacity of the MSC-LV-ShmmiR-129 was 1.8 times that of the MSC cells of the recipient mesenchymal stem cells.

2.3 fluorescent quantitative PCR detection of TNF-alpha and IFN-gamma gene expression levels in C3H10-LV-ShmmiR-129 cells, C3H10-LV-GFP cells and C3H10 cells

Culture solution M1: alpha-MEM containing 10% (by volume) fetal bovine serum and 0. mu.g/mL Lipopolysaccharide (LPS).

Culture solution M2: alpha-MEM containing 10% (by volume) fetal bovine serum and 1.0. mu.g/mL LPS.

Culture solution M3: alpha-MEM containing 10% (by volume) fetal bovine serum and 1.5. mu.g/mL LPS.

Culturing 3 kinds of cells C3H10-LV-ShmmiR-129 cells, C3H10-LV-GFP cells and C3H10 cells infected with the recombinant virus obtained in step 1 for 72 hours in a culture medium M (wherein the culture medium M is a culture medium M1, a culture medium M2 or a culture medium M3) alone at 1.5X 10⁵Per cm²The cells were seeded in 6-well plates at 37 ℃ in 5% CO₂After culturing in the constant temperature incubator for 12h, taking the same number of cells, and carrying out real-time fluorescent quantitative PCR reaction according to the method in the step 2.1 to detect the expression levels of TNF-alpha, IL-10 and iNOS genes in the cells. Wherein, IFN-gamma gene primer:

Forward：5’-GGTCAACAACCCACAGGTC-3’；

Reverse：5’-GACTCCTTTTCCGCTTCCT-3’。

the results showed that the TNF-. alpha.gene expression levels were not significantly different between C3H10-LV-ShmmiR-129 cells, C3H10-LV-GFP cells and C3H10 cells in the same number of cells, both in the treatment with 0. mu.g/mL LPS and in the treatment with 1.0. mu.g/mL LPS; in the same number of cells, the expression level of TNF-alpha gene of C3H10-LV-ShmmiR-129 cells was 1/16 times that of C3H10 cells which are receptor mesenchymal stem cells in the treatment of 1.5. mu.g/mL LPS, and there was no significant difference in the expression level of TNF-alpha gene between C3H10-LV-GFP cells and C3H10 cells.

The same number of cells, IFN-gamma gene expression levels did not differ significantly between C3H10-LV-ShmmiR-129 cells, C3H10-LV-GFP cells and C3H10 cells in 0. mu.g/mL LPS treatment and 1. mu.g/mL LPS treatment; in the same number of cells, the expression amount of IFN-gamma gene of C3H10-LV-ShmmiR-129 cells was 1/14 times that of C3H10 cells which are receptor mesenchymal stem cells in the treatment of 1.5. mu.g/mL of LPS, and there was no significant difference in the expression amount of TNF-alpha gene between C3H10-LV-GFP cells and C3H10 cells.

2.4 detection of nitric oxide secretion Capacity of C3H10-LV-ShmmiR-129 cells, C3H10-LV-GFP cells and C3H10 cells

The 3 cells, C3H10-LV-ShmmiR-129 cells, C3H10-LV-GFP cells and C3H10 cells, which were infected with the recombinant virus obtained in step 1 for 72 hours, were each separately used in culture medium N (culture medium)N is culture solution 1, culture solution 2, culture solution 3), and is cultured at (1-1.5) × 10⁵Per cm²The cells were seeded in 6-well plates at 37 ℃ in 5% CO₂After 12h incubation in the incubator, the supernatant was taken and NO was determined using Griess reagent (Sigma-Aldrich). In short, all NO₃Conversion to NO by NO reductase₂And total NO₂Detection was by Griess reaction.

Culturing 3 kinds of cells, namely C3H10-LV-ShmmiR-129 cells, C3H10-LV-GFP cells and C3H10 cells, which are infected with the recombinant virus obtained in the step 1 for 72 hours, respectively in a culture solution M (the culture solution M is a culture solution M1, a culture solution M2 or a culture solution M3) independently according to the conditions of (1-1.5) × 10⁵Per cm²The cells were seeded in 6-well plates at 37 ℃ in 5% CO₂After 12h incubation in the incubator, the supernatant was taken and NO was determined using Griess reagent (Sigma-Aldrich). In short, all NO₃Conversion to NO by NO reductase₂And total NO₂Detection was by Griess reaction.

The results showed that, in the same number of cells, in the treatment of 0ng/mL of TNF-. alpha.and 0ng/mL of IFN-. gamma.the content of NO in the supernatant of C3H10-LV-ShmmiR-129 cells was 2.50. mu.M, the content of NO in the supernatant of C3H10-LV-GFP cells was 1.60. mu.M, the content of NO in the supernatant of C3H10 cells was 1.56. mu.M, and the NO-secreting ability of C3H10-LV-ShmmiR-129 was 1.6 times that of C3H10 cells which are mesenchymal stem cells of the recipient; the same number of cells, in the treatment of 5ng/mL of TNF-alpha and 5ng/mL of IFN-gamma, the content of NO in the supernatant of C3H10-LV-ShmmiR-129 cells was 13.90. mu.M, the content of NO in the supernatant of C3H10-LV-GFP cells was 7.10. mu.M, the content of NO in the supernatant of C3H10 cells was 7.09. mu.M, and the NO-secreting ability of C3H10-LV-ShmmiR-129 was 2.0 times that of C3H10 cells of mesenchymal stem cells of the recipient; in the same number of cells, the content of NO in the supernatant of C3H10-LV-ShmmiR-129 cells was 19.50. mu.M, the content of NO in the supernatant of C3H10-LV-GFP cells was 10.00. mu.M, the content of NO in the supernatant of C3H10 cells was 10.00. mu.M, and the NO-secreting ability of C3H10-LV-ShmmiR-129 was 2.0 times that of C3H10 cells, which were mesenchymal stem cells, in the treatment of 10ng/mL of TNF-. alpha.and 10ng/mL of IFN-. gamma..

It was revealed that the same number of cells, in the treatment of LPS of 0. mu.g/mL, the content of NO in the supernatant of C3H10-LV-ShmmiR-129 cells was 5.30. mu.M, the content of NO in the supernatant of C3H10-LV-GFP cells was 2.80. mu.M, the content of NO in the supernatant of C3H10 cells was 2.78. mu.M, and the NO-secreting ability of C3H10-LV-ShmmiR-129 was 1.9 times that of C3H10 cells which are mesenchymal stem cells of the recipient; the same number of cells, in the treatment of 1.0. mu.g/mL of LPS, the content of NO in the supernatant of C3H10-LV-ShmmiR-129 cells was 14.00. mu.M, the content of NO in the supernatant of C3H10-LV-GFP cells was 7.30. mu.M, the content of NO in the supernatant of C3H10 cells was 7.36. mu.M, and the NO-secreting ability of C3H10-LV-ShmmiR-129 was 1.9 times that of C3H10 cells which are mesenchymal stem cells of the recipient; the same number of cells, in the treatment with 1.5. mu.g/mL of LPS, the content of NO in the supernatant of C3H10-LV-ShmmiR-129 cells was 19.80. mu.M, the content of NO in the supernatant of C3H10-LV-GFP cells was 10.50. mu.M, the content of NO in the supernatant of C3H10 cells was 10.42. mu.M, and the NO-secreting ability of C3H10-LV-ShmmiR-129 was 1.9 times that of C3H10 cells, which are mesenchymal stem cells of the recipient.

Example 2MSC-LV-ShmmiR-129 attenuated inflammatory injury in mice over recipient mesenchymal stem cells

In order to detect the regulation effect of MSC-LV-ShmmiR-129 on the immune system in an in vivo environment, a model of inflammatory enteritis of mice (a mouse model of ulcerative colitis caused by sodium dextran sulfate) is established by utilizing sodium dextran sulfate (DSS), and the influence of MSCs on the immune system is detected on the basis. The specific method comprises the following steps:

healthy adult C57BL/6 mice, 6 weeks old, 22-25g, were housed in the center of the military medical academy of sciences laminar flow purge animals on a common diet for one week prior to the experiment. The experiment was divided into 4 groups of 5: a negative control group, a positive control group, an MSC-LV-GFP group and an MSC-LV-ShmmiR-129 group. The day on which the test started was recorded as day 0. On day 0, body weight was weighed. Each mouse in the negative control group was fed with sterile water daily, and each mouse in the three groups of the positive control group, MSC-LV-GFP group and MSC-LV-ShmmiR-129 group was fed with a sterile aqueous solution of 5% dextran sodium sulfate daily. Day 1, intraperitoneal injection was performed on each mouse in the two groups of the negative control group and the positive control groupPBS, MSC-LV-GFP group each mouse was injected intraperitoneally with an equal volume of 1X 10⁶Cell suspensions of individual MSC-LV-GFP (prepared with PBS and MSC-LV-GFP), MSC-LV-ShmmiR-129 groups of mice each injected intraperitoneally with an equal volume of 1X 10⁶Cell suspensions of MSC-LV-ShmmiR-129 (formulated with PBS and MSC-LV-ShmmiR-129). The water feeding is changed at regular time every day. Mice were observed daily for posture, hair, stool morphology, and presence or absence of hematochezia. Disease index (pathology index) and body weight were recorded, and the score criteria for pathology index are shown in table 1. On day 8, mice were sacrificed. After dissection, the cecum, mesenteric lymph nodes and peripheral blood were taken. The mouse cecal length, cecal weight and spleen weight were also weighed.

TABLE 1 pathological index scoring criteria

Score value	Body weight	Stool form	Presence or absence of hematochezia
				0	Without loss (compared with negative control group)	Normal (compared with negative control group)	Normal (compared with negative control group)
1	<5％	Partial dryness	Slight redness
				2	5％-10％	Hard and dry	Tan color
3	More than 10 percent to less than or equal to 15 percent	Loose (formable)	Occult blood (with spot blood silk)
				4	More than 15 percent to less than or equal to 20 percent	Dilute (formable)	Obvious blood
5	>20％	Diarrhea (unshaped)	Severe hemorrhage

The results showed that the positive control group showed stool dilution and hematochezia earlier than the MSC-LV-GFP group and the MSC-LV-ShmmiR-129 group, and the MSC-LV-GFP group showed stool dilution and hematochezia earlier than the MSC-LV-ShmmiR-129 group. After miR-129 expression is interfered, compared with MSC-LV-GFP cells, the MSC-LV-ShhmiR-129 cells with the same quantity can obviously improve pathological indexes of a colitis model mouse, obviously relieve weight loss of the mouse, obviously increase colon weight, colon length, spleen weight and the like, and obviously inhibit development of colitis conditions of the mouse (figures 1-3).

IBD models were used to detect differences in the immunomodulatory capacity of the MSC-LV-GFP group and MSC-LV-ShmmiR-129 in vivo. The in vivo IBD model demonstrated that MSC-LV-ShmmiR-129 has a greater ability to attenuate inflammatory lesions in mice than MSC-LV-GFP.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> military medical research institute of military science institute of people's liberation force of China

<120> mesenchymal stem cell for efficiently inhibiting immune reaction and preparation method and application thereof

<130> GNCFH182386

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> RNA

<213> mouse (Mus musculus)

<400> 1

cuuuuugcgg ucugggcuug c 21

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gcaagcccag accgcaaaaa g 21

<210> 3

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gcaagcccag accgcaaaaa g 21

Claims

1. A preparation method of recombinant mesenchymal stem cells comprises the following steps: reducing the expression quantity of microRNA-129 genes in the receptor mesenchymal stem cells to obtain recombinant mesenchymal stem cells; the receptor mesenchymal stem cell is an in vitro mesenchymal stem cell.

2. The method of claim 1, wherein: the nucleotide sequence of the microRNA-129 is SEQ ID No. 1.

3. The production method according to claim 1 or 2, characterized in that: the method for reducing the expression quantity of the microRNA-129 gene in the receptor mesenchymal stem cell comprises the step of introducing a substance for inhibiting the expression of the microRNA-129 gene into the receptor mesenchymal stem cell.

4. The production method according to claim 1 or 2, characterized in that:

the recombinant mesenchymal stem cells have at least one of the following characteristics A1) -A5):

A3) the expression capacity of proinflammatory factors of the recombinant mesenchymal stem cell is lower than that of the receptor mesenchymal stem cell;

A4) the recombinant mesenchymal stem cell has higher anti-inflammatory factor expression capacity than the receptor mesenchymal stem cell;

5. The production method according to claim 3, characterized in that: the substance for inhibiting the expression of the microRNA-129 gene is B1) or B2):

B1) RNA of which the nucleotide sequence is SEQ ID No. 3;

B2) expression vector for expressing the RNA of B1).

6. The production method according to claim 1 or 2, characterized in that: the isolated mesenchymal stem cell is an isolated mesenchymal stem cell of a mammal.

7. The recombinant mesenchymal stem cell prepared by the preparation method of any one of claims 1-6.

8. Any of the following applications:

use of P1, the recombinant mesenchymal stem cell of claim 7, in the preparation of an immunosuppressive product;

use of P2, the recombinant mesenchymal stem cell of claim 7, in the manufacture of a product for attenuating inflammatory injury;

p3, the use of the substance for inhibiting the expression of the microRNA-129 gene in the preparation method of claim 5 in the preparation of a product for enhancing the nitric oxide secretion capability of mesenchymal stem cells;

p4, the use of the substance for inhibiting the expression of the microRNA-129 gene in the preparation method of claim 5 in the preparation of a product for enhancing the immunosuppressive capacity of mesenchymal stem cells;

p5, the use of the substance for inhibiting the expression of the microRNA-129 gene in the preparation method of claim 5 in the preparation of a product for reducing the expression capacity of proinflammatory factors of mesenchymal stem cells;

p6, the substance for inhibiting the microRNA-129 gene expression in the preparation method of claim 5, and the application thereof in preparing products for enhancing the anti-inflammatory factor expression capability of mesenchymal stem cells.