CN104800243B

CN104800243B - Application of recombinant mesenchymal stem cells in preparation of immunosuppressant

Info

Publication number: CN104800243B
Application number: CN201410188453.1A
Authority: CN
Inventors: 李红; 郭希民; 毛宁; 江小霞
Original assignee: Institute of Basic Medical Sciences of AMMS
Current assignee: Institute of Basic Medical Sciences of AMMS
Priority date: 2014-01-28
Filing date: 2014-05-06
Publication date: 2021-04-30
Anticipated expiration: 2034-05-06
Also published as: CN104800243A

Abstract

The invention discloses an application of a recombinant mesenchymal stem cell in preparation of an immunosuppressant. The recombinant mesenchymal stem cells can be subjected to targeted migration in vivo to a secondary lymphoid organ after being infused and can be massively accumulated in a T cell enrichment area in the secondary lymphoid organ, the immunoreaction of T cells can be effectively inhibited, and the cost, the cell dosage and the side effect can be reduced in the treatment of graft-versus-host disease after bone marrow transplantation, immune rejection reaction after organ transplantation and autoimmune diseases. More particularly, the effect of killing tumor cells of the organism is kept in the treatment, and a high-efficiency and beneficial new strategy is provided for clinically treating graft-versus-host disease after bone marrow transplantation, immune rejection after organ transplantation and autoimmune disease of high-risk tumor people.

Description

Application of recombinant mesenchymal stem cells in preparation of immunosuppressant

Technical Field

The invention relates to the field of stem cell therapy, in particular to application of a mesenchymal stem cell of which the cell membrane expresses a chemokine receptor CCR7 in preparation of an immunosuppressant.

Background

Mesenchymal stem cells are a population of adult stem cells with self-renewal and multipotent differentiation potential that were first isolated from bone marrow. Recent studies have shown that: mesenchymal stem cells have a wide range of immunomodulatory functions. The mesenchymal stem cells have the immunoregulation function on various immune cells including T cells, B cells, NK cells, DCs and the like. In effective animal experiments and clinical disease treatment, mesenchymal stem cells are found to play a role in vivo immunoregulation mainly through secretory cytokines.

Currently, the immunotherapy of clinical diseases such as hematopoietic stem cell transplantation or graft-versus-host disease, immune rejection after solid organ transplantation, crohn's disease, systemic lupus erythematosus and the like by mesenchymal stem cells has entered the clinical phase II-III in the united states. 5/17/2012, the health regulatory agency in Canada announced approval of a human mesenchymal stem cell drug, procymmal^TMMarketed under the condition of allowing Prochymal^TMBecomes the first stem cell medicine which is really approved to be used for treating the systemic diseases in the world, and marks that the door of the clinical application of the mesenchymal stem cells as the immunomodulator is opened. The problems still faced are: 1) phase III clinical results in multicenter indicate: the in vivo immunoregulation effect is not obvious; 2) large cell usage [ Tyndall a. vaccines and failures of stem cell transfer in autoimmune diseases. hematology Am Soc Hematol Educ program.2011; 2011: 280-284.][Martin I，Baldomero H，Tyndall A，Niederwieser D，Gratwohl A.A survey on cell and engineered tissue therapies in Europe in2008.Tissue Eng Part A2010；16：2419-2427.]

Many clinical treatment modalities of mesenchymal stem cells include in situ injection and intravenous injection [ http: // www.clinicaltrials.gov `]. However, intravenous injection is simple and easy, and systemic diseases such as graft-versus-host disease, Crohn's disease, systemic lupus erythematosus and the like are mainly intravenous injection. Therefore, a large amount of cells are needed for clinically treating the diseases, for example, the clinical mesenchymal stem cell immunotherapy dosage is 1-9 x 10⁶MSCs/Kg, 2 times per week for four weeks, clearly increases the cost of treatment, and may also present risks such as embolism, tumor recurrence, etc. Thus, mesenchymal stem cells are efficientIn vivo migration is the key to achieving the two most efficient and economical goals of Cell therapy [ Karp JM and Leng Teo gs.mesenchyme Stem Cell hosting: the Devil Is in The details cell Stem cell.2009; 4(3): 206-216.Ankrum J, Karp JM.Mesenchyl stem cell therapy: two steps forward, one step back. trends Mol med.2010; 16(5): 203-9]。

Secondary lymphoid organs including spleen white marrow (SPW), Lymph Nodes (LN), Mesenteric Lymph Nodes (MLN), PP nodes, etc., are simple and similar in structure, and are composed of T, B lymphocytes, Antigen Presenting Cells (APC), stromal cells, and microtubule junctions. T cells receive antigen presented by APC in secondary lymphoid organs, and proliferate by activation, and thus, secondary lymphoid organs are the sites where immune responses are initiated in vivo. After intravenous infusion, mesenchymal stem cells are widely distributed in the whole body, such as lung, intestine, liver, bone marrow, and the like, but rarely exist in secondary lymphoid organs. Mesenchymal stem cells are "diluted" in almost all tissues and organs, making them simple and thin in the upper body of the main battlefield, so that they have strong immunoregulatory capacity in vitro experiments, but have unsatisfactory effects in vivo immunoregulatory therapy.

Clinical tumor patients may be at increased risk of tumor recurrence following hematopoietic stem cell transplantation, such as by massive mesenchymal stem cell infusion [ Ning H, the clinical study of metabolic stem cells and high recurrence rates in clinical malignity patents: outcome of a pilot clinical study. Leukemia.2008; 22: 593-599]. And a large number of research results show that Mesenchymal Stem Cells can promote the generation, growth, drug resistance, invasion and metastasis of tumors [ Eterno V, et al. adopise-derived sensory Stem Cells (ASCs) may facial nerve cancer cell VIF/c-Met signaling. oncotarget.2013; epub ahead of print ] [ Bergfeld SA, Blavier L, Declerck YA. bone Marrow-Derived Mesenchyl structural Cells project overview and Drug Resistance in Tumor Cells The.2014; epub ahead of print ] [ Wang D1, Wang S, Shi C.update on placed diseases of sensory Stem Cell-based therapeutics. Current Stem Cell Res. 2012; 7(5): 370-80.].

In clinical treatment of graft-versus-host disease after hematopoietic stem cell transplantation of tumor patients, immune rejection after organ transplantation in high risk groups with tumor occurrence and autoimmune disease treatment, for example, application of mesenchymal stem cell mass infusion with wide immune suppression function, except for the defects of high price and influence on blood homeostasis, the larger problem is that the mesenchymal stem cell mass infusion brings the risk of increasing tumor occurrence/recurrence. Therefore, common mesenchymal stem cells are cautious, even forbidden, in the treatment of these groups of people.

Based on the disadvantages of weak in-vivo immune regulation capability, large cell dosage and potential increase of tumor recurrence/occurrence risk of common mesenchymal stem cells in clinical treatment, the demand of more economical and efficient cell treatment strategies capable of overcoming the disadvantages is particularly urgent.

Disclosure of Invention

The invention provides an application of a recombinant mesenchymal stem cell in preparation of an immunosuppressant. By modifying the mesenchymal stem cells, the cell membranes of the mesenchymal stem cells express CCR7, the mesenchymal stem cells after infusion are guided to migrate in a targeting manner to enter a secondary lymphoid organ and can be contacted with T lymphocytes in a short distance in a T cell enrichment area gathered in the secondary lymphoid organ, and the T cell mediated immune reaction is effectively inhibited. More particularly, the recombinant mesenchymal stem cells of the invention simultaneously retain the anti-tumor capacity of the body.

In order to achieve the purpose, the invention comprises the following technical scheme:

the application of the recombinant mesenchymal stem cells in preparing the immunosuppressant is characterized in that the recombinant mesenchymal stem cells are obtained by expressing a chemokine receptor CCR7 on the cell membrane of the mesenchymal stem cells, and the immunosuppressant is applied to graft-versus-host disease after hematopoietic stem cell transplantation of high risk tumor groups, immune rejection reaction and/or autoimmune disease after organ transplantation.

For use as described above, preferably the mesenchymal stem cells are of autologous, allogeneic or commercial origin.

The use as described above, preferably, the method for expressing the chemokine receptor CCR7 on the cell membrane of the mesenchymal stem cell comprises: introducing a CCR7 gene into the mesenchymal stem cell by adopting a gene transfection method, so that the cell membrane of the mesenchymal stem cell expresses CCR 7; expressing CCR7 through a vector transfection system on a mesenchymal stem cell membrane; using Trolocytosis method, using antigen presenting cell to present CCR7 to the cell membrane of mesenchymal stem cell; or by modification of existing cell membrane proteins or by exoproteins.

The gene transfection method comprises a viral vector transfection system and a non-viral transfection system.

As mentioned above, preferably, the viral vector transfection system is any one or more of adeno-associated virus AA-V, lentivirus, adenovirus Ad-V, baculovirus, herpes virus and retrovirus transfection system.

As mentioned above, preferably, the non-viral transfection system is a plasmid vector transfection system, a chitosan nanoparticle transfection system, or a naked gene transfection system.

The use as described above, preferably, the high risk group of tumors comprises: 1) middle aged and elderly people over 45 years old; 2) high risk group caused by genetic factors: the history that the direct or collateral relatives within the third generation suffer from malignant tumor; 3) people with bad life habits: people who smoke abundantly for a long time, drink abuses for a long time, abuse of drugs, long-term overwork, stay up all night, have high pressure of life, high pressure of work, have bad mood for a long time, have depressed characters or use the mobile phone frequently; 4) people with poor eating habits: food preference, malnutrition, unclean diet, irregular diet, slow eating speed, frequent eating of overheated, hard and spicy food or frequent eating of pickled smoked fried food; 5) people with occupational factors exposed to toxic and harmful substances for a long time; 6) the people with polluted living environment: water source and/or air chemical pollution, heavy metal pollution, nuclear pollution, microbial pollution or people living in haze weather for a long time; 7) people with chronic or recurrent inflammation: chronic bronchitis, chronic hepatitis B virus infected person, AIDS virus infected person, chronic cervicitis, chronic gastritis, stomach polyp, gastric ulcer, mastitis, mammary gland chronic hyperplasia, intestinal polyp, gallbladder polyp, prostatitis, sinusitis or long-term functional uterine bleeding; 8) and treating benign and malignant tumor patients.

The use as described above, preferably, the autoimmune disease is systemic lupus erythematosus, xerostomia syndrome, rheumatoid arthritis, ankylosing spondylitis, scleroderma, polyarteritis nodosa, Wegener's granulomatosis, ulcerative colitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, dermatomyositis, mixed connective tissue disease, myasthenia gravis, thyroid autoimmune disease, insulin-dependent diabetes mellitus, malignant anemia with chronic atrophic gastritis, goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis or acute idiopathic polyneuritis.

For use as described above, preferably, the recombinant mesenchymal stem cells are used in combination with a conventional immunosuppressive agent.

For the above applications, preferably, the dosage of the recombinant mesenchymal stem cells to human body is 1 × 10⁴～1×10⁶MSCs/kg。

The invention has the beneficial effects that:

1. the cell membrane of the mesenchymal stem cell expresses CCR7, after being infused, the mesenchymal stem cell can target the migration of a secondary lymph organ in vivo, and can be greatly accumulated in the local part of the secondary lymph organ, particularly the mesenchymal stem cell can be accumulated in a T cell enrichment area of the secondary lymph organ and can be in close contact with T lymphocytes, so that the T cell mediated immune reaction is effectively inhibited, the cell dosage can be reduced when the graft-versus-host disease after hematopoietic stem cell transplantation, the immune rejection reaction after solid organ transplantation and the autoimmune disease are clinically treated, the cost is reduced, and the side effects of embolism and the like are reduced.

2. The recombinant mesenchymal stem cells of the invention simultaneously reserve the anti-tumor effect of the organism. Therefore, the cell can reduce the risk of tumor recurrence when clinically treating the graft-versus-host disease after the hematopoietic stem cell transplantation of a tumor patient; the risk of tumorigenesis can also be reduced in the treatment of autoimmune diseases and immune rejection reactions after organ transplantation in the high risk group of tumorigenesis.

The immunosuppressant prepared by the recombinant mesenchymal stem cell overcomes the defects of weak in-vivo immunoregulation capability, large cell dosage and potential increase of tumor recurrence/occurrence danger of the common mesenchymal stem cell when treating high-incidence immune reaction of clinical patients, and is obviously superior to the common mesenchymal stem cell. The invention provides a novel efficient and beneficial strategy for clinically inhibiting the immune response of a patient.

Drawings

FIG. 1: CCR7 expression status of Mesenchymal Stem Cells (MSCs).

A: detecting the CCR7 expression conditions of Balb/C mice and C57BL/6 mouse bone marrow-derived MSCs by an RT-PCR method;

b: detecting the expression of eGFP and CCR7 of the three cells by an RT-PCR method;

c: detecting the mRNA expression level of the eGFP genes of the three cells by real-time quantitative PCR;

d: detecting the mRNA expression level of the CCR7 genes of the three cells by real-time quantitative PCR;

e: flow cytometry detected the membrane expression of CCR7 in three cells.

FIG. 2A: fluorescence microscopy images of three groups of mice Spleen (SP), Lymph Node (LN), Mesenteric Lymph Node (MLN), PP node (PP) sections with DAPI nuclear markers.

FIG. 2B: eGFP⁺Frequency of MSCs in various tissues and organs in vivo.

FIG. 3: MSCs/CCR7-eGFP post tail vein infusion in spleen and lymph nodes with CD3⁺T cells, CD11c⁺Dendritic cells and B220⁺Photomicrographs of B cells.

FIG. 4: experimental results of MSCs/CCR7-eGFP for highly inhibiting graft versus host disease (GvHD).

FIG. 5: in GvHD mouse model, MSCs/CCR7-eGFP was tested for the ability to resist tumors in the body.

FIG. 6: in a mouse xenogeneic skin transplantation model, the results of the immune regulation and control of the MSCs/CCR7-eGFP and the test of the influence on the growth capacity of the tumor of the organism are shown.

FIG. 7: the results of the immune regulation of MSCs/CCR7-eGFP and the test of the influence on the growth capacity of the tumors in the body in a mouse inflammatory bowel disease model.

Detailed Description

The expression of the chemokine receptor CCR7 on the cell membrane of the mesenchymal stem cell can be realized by the following method: introducing a CCR7 gene into the mesenchymal stem cell by adopting a gene transfection method, so that the cell membrane of the mesenchymal stem cell expresses CCR 7; expressing CCR7 through a vector transfection system on a mesenchymal stem cell membrane; using Trolocytosis method, using antigen presenting cell to present CCR7 to the cell membrane of mesenchymal stem cell; or by modification of existing cell membrane proteins or by exoproteins.

Wherein, the gene transfection method comprises a viral vector transfection system and a non-viral transfection system; the virus vector transfection system can be any one or more of adeno-associated virus AA-V, lentivirus, adenovirus Ad-V, baculovirus, herpes virus and retrovirus transfection systems; the non-viral transfection system can be a plasmid vector transfection system, a chitosan nanoparticle transfection system or a naked gene transfection system.

The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples.

In the following examples, unless otherwise specified, all methods are conventional.

Example 1 preparation of recombinant mesenchymal Stem cells expressing chemokine receptor CCR7

1. Mouse bone marrow MSCs separation culture

The sample is 2-3 weeks old C57BL/6 mouse (Experimental animal center of military medical science institute), the mouse is killed by breaking neck, the mouse is soaked in 75% alcohol and disinfected → the tibia and femur of the mouse are separated under aseptic condition, the mouse is placed in a flat dish → the adhered muscle is rubbed by using sterile gauze, the metaphysis is removed → the marrow cavity is repeatedly washed by PBS + 10% FCS (Hyclone), the marrow cells are washed out → inoculated in a culture bottle, the bone fragments are uniformly distributed on the bottom of the bottle, the culture system contains alpha t-MEM (Gibco) + 10% FCS (Hyclone) +100U/ml penicilliumSumin +100U/ml streptomycin, 37 deg.C, 5% CO₂Culturing under saturated humidity conditions → changing the solution 1 time every 3 days → when the cells are approximately 70-80% confluent, digesting and passaging with 0.25% pancreatin (Hyclone), and the inoculation density of the passaged cells is 4000 cells/cm²。

2. Adeno-associated virus infected mouse MSCs

eGFP-adeno-associated virus vector and CCR 7-eGFP-adeno-associated virus vector, both of which were prepared by Invitrogen, were transferred to mouse MSCs. The method comprises the following steps: spreading MSCs on 48-well plate (2 × 10)⁴A hole. The next day, the medium was changed to a new medium, to which virus (MOI ═ 20), polybrene (8 μ g/ml) was added, and the normal culture system was changed 6 hours after infection. The MSCs introduced with the eGFP are named MSCs/eGFP; MSCs introduced into the CCR7-eGFP vector were designated MSCs/CCR 7-eGFP.

RT-PCR detection of CCR7 expression of MSCs at mRNA level

MSCs isolated from BalB/C mice and C57BL/6 mice were collected, splenic mononuclear cells (SPMNC) of the same strain were used as positive controls, and transfected MSCs/eGFP, MSCs/CCR7-eGFP, were collected. The RT-PCR method is used for detecting the mRNA level of the cells expressing CCR 7. The primers are as follows:

TABLE 1

The results are shown in FIG. 1, FIG. 1A: RT-PCR results showed that neither BalB/C mice nor C57BL/6 mouse-derived MSCs expressed CCR7, whereas spleen cells of the same strain origin (SPMNC) were positive. The expression of housekeeping gene HPRT is used as an internal reference; FIG. 1B: normal MSCs were used as blank controls, MSC with empty vector as transfection control (MSCs/eGFP), and CCR7 gene was transferred into MSCs (MSCs/CCR 7-eGFP). The RT-PCR detection result shows that: MSC/eGFP and MSC/eGFP-CCR7 express eGFP; of the three cells, MSC/CCR7-eGFP successfully expressed CCR7 mRNA, and expression of the housekeeping gene HPRT served as an internal control. This result indicates that the CCR7 gene was successfully transferred into MSCs.

4. Real-time quantitative PCR method for detecting mRNA level of expression CCR7 of MSCs

And respectively collecting three groups of cells including MSCs, MSCs/eGFP and MSCs/eGFP-CCR 7. RNA was extracted by the conventional method and the mRNA level of CCR7 of the three cells was detected by real-time quantitative PCR. The primers are as follows:

TABLE 2

See fig. 1C, D: detecting mRNA expression levels of eGFP genes and CCR7 genes of three cells including MSCs, MSCs/eGFP and MSCs/eGFP-CCR7 by real-time quantitative PCR; the results of fig. 1B are further confirmed.

5. Flow cytometry detection of MSCs membrane expression CCR7

The three groups of cells, namely the MSCs, the MSCs/eGFP and the MSCs/CCR7-eGFP, are respectively blocked by a mouse CD16/32 antibody (ebioscience company), and then the cells are labeled by a CCR7-PE antibody (ebioscience company), and the detection result of a flow cytometer in FIG. 1E shows that: the MSCs and the MSCs/eGFP do not express the membrane molecule CCR7, and 93.0-98.4% of the MSCs/CCR7-eGFP expresses the membrane molecule CCR 7.

Example 2 detection of the ability of MSCs to migrate to Secondary lymphoid organs in vivo and to enrich the region

1. Detection of migration ability of MSCs to secondary lymphoid organs in vivo

Infusion of MSCs, MSCs/eGFP or MSCs/CCR7-eGFP (1X 10)⁶) Three groups of mice (4 per group); spleen (SP), Lymph Node (LN), Mesenteric Lymph Node (MLN), PP node were cryosectioned 5 days after infusion and nuclear labeling was performed with DAPI. The green fluorescent cells were observed under a fluorescence microscope. The results in FIG. 2A show that: MSCs/eGFP did not have the ability to migrate to secondary lymphoid organs, whereas MSCs/eGFP-CCR7 has a large distribution in SP, LN, MLN, PP. FIG. 2B shows the calculation of eGFP for each organ⁺The frequency of distribution of MSCs in each tissue organ in vivo (number of green fluorescences/high power field) quantitatively showed that infused MSCs/eGFP had no ability to migrate to secondary lymphoid organs in vivo, whereas infused MSCs/eGFP-CCR7 had the ability to target migration of secondary lymphoid organs in vivo.

MSCs/eGFP-CCR7 present in T cell-rich regions in secondary lymphoid organs

Mixing MSCsFrozen sections of SP, LN from mice infused with/eGFP-CCR 7 were immunofluorescent stained and incubated with phycoerythrin-labeled (PE) CD3 or B220 antibodies and then observed under a fluorescent microscope for MSCs/CCR7-eGFP (green) and CD3⁺T lymphocytes (Red, FIG. 3A), CD11c⁺Dendritic cells (Red, FIG. 3B) or B220⁺Relationship of B lymphocytes (red, fig. 3C), DAPI (blue) staining shows nuclei. There are many cells in the secondary lymphoid organs, MSCs/CCR7-eGFP are present in T cell-enriched regions in secondary lymphoid organs, which lays the foundation for MSCs/CCR7-eGFP having strong immunomodulatory capacity in vivo against T cell-mediated immune responses.

Example 3 detection of the Effect of MSCs/CCR7-eGFP on in vivo immunomodulation and Effect on tumor growth

Inhibition of GvHD by MSCs/CCR7-eGFP

GvHD is a typical T cell-mediated immune disease. Establishing a mouse GvHD model: collecting 20-24g BALB/c male mouse, collecting spleen cells and bone marrow cells, and counting; 2X 10⁷Splenocytes +1 × 10⁷Bone marrow cells/0.25 ml PBS tail vein injection of 22-24g C57BL/6 male mice irradiated with 9Gy to establish aGvHD model (GvHD group). On the basis of the above-mentioned two-step co-infusion, 1X 10⁵Or 1X 10⁶ 1X 10 MSCs/eGFP of⁵ Or 1X 10⁶MSCs/CCR7-eGFP。1×10⁶Mice co-infused with MSCs/eGFP tail vein were referred to as GvHD + MSCs/eGFP mice; 1X 10⁶Mice co-infused tail vein with MSCs/CCR7-eGFP were designated GvHD + MSCs/CCR7-eGFP mice.

After infusion, three groups of mice were observed daily for survival and status, survival curves were recorded to observe the in vivo immunomodulatory effects of MSCs, and serum cytokine levels were measured. As shown in FIG. 4A, the MSCs/CCR7-eGFP infusion significantly prolonged the survival time of GvHD mice, with effects significantly better than the same dose of MSCs/eGFP infusion group. After 2 weeks of modeling, GvHD mice showed weight loss, bows and backs, diarrhea, rough hair, and reduced mobility. The GvHD performance of mice infused with MSCs/CCR7-eGFP is obviously improved, and the effect of the GvHD is obviously better than that of the MSCs/eGFP co-infusion group with the same dose (figure 4B). ELISA results showed that co-infusion of MSCs/CCR7-eGFP significantly increased the level of anti-inflammatory factor IL-4 in the serum of mice (FIG. 4C) and significantly decreased the level of inflammatory factor IFN-. gamma. (FIG. 4D) compared to GvHD, GvHD + MSCs/eGFP mice. The above results demonstrate that: MSCs expressing CCR7 have greater in vivo immunomodulatory capacity than normal MSCs.

2. Detection of the influence of MSCs/eGFP-CCR7 on the anti-tumor capability of an organism in a mouse GvHD model

Mouse GvHD, GvHD + MSCs/eGFP (1X 10)⁶)、MSCs/CCR7-eGFP(1×10⁶) On the day of model creation, co-infusion was 5 × 10⁵EL4 leukemia cells to establish a tumor-bearing GvHD mouse model. Survival time of each group of mice was recorded, survival curves were plotted, and the effect of the two MSCs on tumor growth was observed. As a result, it was found that: MSCs/eGFP co-infusion did not prolong the survival time of mice compared to GvHD + EL4 tumor-bearing mice, whereas MSCs/eGFP-CCR7 co-infusion significantly prolonged the survival time of mice (fig. 5A); tumor infiltration of the liver of each group of mice was observed 2 weeks after modeling, and the results in fig. 5B show: syngeneic splenocytes and tumor cells co-infused group (syno-infusion + EL4) mice had obvious tumor nodules in their livers, demonstrating successful tumor loading in mice (FIG. 5 Ba); the liver of mice in GvHD + EL4 group had obvious hemorrhage and necrosis, indicating that the mice had obvious GvHD (figure 5 Bb); the GvHD + EL4+ MSCs/eGFP group mice have certain improvement on liver GvHD change and still have a plurality of tumor nodules (figure 5 Bc); the GvHD + EL4+ MSCs/CCR7-eGFP mice have improved hemorrhage, necrosis and tumor nodules, which indicates that the co-infusion of the MSCs/CCR7-eGFP reduces GvHD and simultaneously retains the anti-tumor capability of the body (figure 5 Bd);

3. xenograft skin graft model (alloSG) mice were loaded with melanoma (B16) and the effect of MSCs/eGFP-CCR7 on the ability of body tumor growth was examined.

Establishing a melanoma-loaded xenogeneic skin transplantation mouse model (alloSG + B16+ PBS): Balb/C mice skin (1 Gm)²) Transplanted to the back of C57 mouse, melanoma cells (B16) 5X 10⁵Performing lateral abdominal subcutaneous injection, establishing a melanoma-loaded mouse xenogeneic skin transplantation model, and infusing 0.2ml PBS (phosphate buffer solution) into tail vein on the next day; tail vein infusion of 1X 10 on model basis⁶MSCs/eGFP, defined as alloSG + B16+ MSCs/eGFP set; or tail vein infusion of 1X 10⁶MSCs/CCR7-eGFP, defined as alloSG + B16+ MSCs/CCR7-eGFP group.

The survival time of the xenogenic skin of the three groups of mice was observed and showed that the xenogenic skin survival time of the mice of the alloSG + B16+ MSCs/CCR7-eGFP group was significantly prolonged (×, p < 0.05), whereas the survival time of the xenogenic skin of the mice of the alloSG + B16+ MSCs/eGFP group was not different from that of the control group (fig. 6A). After 2 weeks of modeling, the melanoma of the alloSG + B16+ MSCs/eGFP group was significantly larger in volume and significantly increased in weight (p < 0.05) than the control group; however, the melanoma group of alloSG + B16+ MSCs/CCR7-eGFP was not significantly different from the control group in both volume and weight (n.s., no significant difference) (fig. 6B, C).

4. In an melanoma-loaded mouse Inflammatory Bowel Disease (IBD) model, the immune regulation of MSCs/CCR7-eGFP and the ability to grow tumors in the body were tested.

Mice were fed with 2% DSS for 7 consecutive days, with melanoma cells (B16) at 5X 10 on the first day of feeding water⁵Lateral abdominal subcutaneous injection, tail vein infusion of 0.2ml PBS induced a melanoma-loaded mouse model of inflammatory bowel disease (IBD + B16). Tail vein infusion of 1X 10 on model basis⁶MSCs/eGFP, defined as IBD + B16+ MSCs/eGFP group; or tail vein infusion of 1X 10⁶MSCs/CCR7-eGFP, defined as the IBD + B16+ MSCs/CCR7-eGFP group.

The survival time of three groups of mice is observed, and the result shows that: IBD + B16+ MSCs/CCR7-eGFP mice survived significantly longer than controls (FIG. 7A). After 14 days of modeling, the melanoma of IBD + B16+ MSCs/eGFP group is obviously larger than that of the control group in volume and obviously increased in weight (p is less than 0.05); however, there was no significant difference in the volumes and weights of the IBD + B16+ MSCs/CCR7-eGFP group melanomas from the control group (n.s., no significant difference) (fig. 7B).

Claims

1. The application of recombinant mesenchymal stem cells in preparing an immunosuppressant for treating immunosuppressive diseases and retaining the anti-tumor effect of an organism is disclosed, the recombinant mesenchymal stem cells are obtained by expressing a chemokine receptor CCR7 on cell membranes of the mesenchymal stem cells, and the immunosuppressive diseases are graft-versus-host diseases after hematopoietic stem cell transplantation, immune rejection reactions after organ transplantation and/or autoimmune diseases.

2. The use of claim 1, wherein the recombinant mesenchymal stem cells are autologous or allogeneic in origin.

3. The use of claim 1, wherein the method of expressing chemokine receptor CCR7 on the cell membrane of a mesenchymal stem cell comprises: introducing a CCR7 gene into the mesenchymal stem cell by adopting a gene transfection method, so that the cell membrane of the mesenchymal stem cell expresses CCR 7; expressing CCR7 through a vector transfection system on a mesenchymal stem cell membrane; using Trolocytosis method, using antigen presenting cell to present CCR7 to the cell membrane of mesenchymal stem cell; or by modification of existing cell membrane proteins or by exoproteins.

4. The use of claim 3, wherein the gene transfection method comprises a viral vector transfection system and a non-viral transfection system.

5. The use according to claim 4, wherein the viral vector transfection system is any one or more of adeno-associated virus AA-V, lentivirus, adenovirus Ad-V, baculovirus, herpes virus and retrovirus transfection systems.

6. The use of claim 4, wherein the non-viral transfection system is a plasmid vector transfection system, a chitosan nanoparticle transfection system, or a naked gene transfection system.

7. The use of claim 1, wherein the tumor is melanoma.

8. The use as claimed in claim 1, wherein the autoimmune disease is systemic lupus erythematosus, xerostomia syndrome, rheumatoid arthritis, ankylosing spondylitis, scleroderma, polyarteritis nodosa, Wegener's granulomatosis, ulcerative colitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, dermatomyositis, mixed connective tissue disease, myasthenia gravis, thyroid autoimmune disease, insulin-dependent diabetes mellitus, pernicious anemia with chronic atrophic gastritis, goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis of the cerebrospinal fluid or acute idiopathic polyneuritis.

9. The use of any one of claims 1-8, wherein the recombinant mesenchymal stem cells are used in combination with a conventional immunosuppressive agent.