| Objective:1. To establish a method for isolation and culture of bone marrow mesenchymal stem cells(MSCs) from Lewis rat in vitro and to identify characteristic of the cells after culture expansion.2. To study probability of experimental autoimmune myasthenia gravis with rat 97-116(Rα97-116) peptide of the acetylcholine receptor(AchR)α-subunit induced and provide a simple and available animal models for basic researches of MG.3. To study the effect of marrow mesenchymal stem cells(MSCs) on the clinical manifestation and the immunity function of EAMGMethods:MSCs were obtained from Lewis rats and planted in plastic culture capsule,then cultured and expanded in vitro.MSCs were identified by flow cytometry. Female Lewis rats(6-8 weeks) were immunized subcutaneously with the synthetic peptide Rα97-116 in CFA and boosted on day 30 and 60 with the same peptide in IFA to iduce rat models of EAMG in experiment group and only with PBS in control group. Clinical manifestation was evaluated by measurement of body weight and Lennon clinical score. Disease was further confirmed by 3,5 Hz repetitive nerve stimulation (RNS) for positive decremental respnse and ELISA assay for serum AchR-ab titers. A week after the third immunization,20 EAMG rats were determined and divided ramdomly into treatment group and control group, treatment group were injected with 106 MSCs diluted in PBS (100μL) or with an equal volume of PBS in control group.And then the body weight and clinical symptoms of both two group were evaluated at the same time every day. The levels of anti-AChR in serum were tested by ELISA after 2 weeks. The expressions of CD28,CTLA4,CD25 molecules on the surface of CD4+T cell in peripheral blood cells were exaimed by flow cytometry in treatment group,control group and healthy rat after 3 weeks.Results:1. After 24 hours primary culture , MSCs adhered to plastic surface of the culture dish.After 48～72 hours culture , the cells proliferated in colonies.These primary MSCs reached 70 %～80 % of confluence in7～10days, 97.5% of the 5 passages of MSCs express CD90 and lack expression (1.72% positive) of CD452. Signs of EAMG occurred in 70% of rats immunized with rat-derived Rα97-116 peptide ,wich was confirmed by the positive decremental response D5 >10% of 3,5 Hz RNS and the ratio >2.1 of the serum AchR-ab titers to control group.The achievement ratio of EAMG was 70%.3. The 15th day after EAMG received MSCs, the body weight of rats in the treatment group were increased significantly compared with the control group(p﹤0.05).4. The 13th day after EAMG received MSCs, the changes of the mean clinical score of rats in the treatment group decreased (p﹤0.05).5. Compared with the control group , the amount of anti-AChR IgG in serum in the treatment group decreased significantly (p﹤0.05) 2 weeks after MSCs Transplantation.6. 3 weeks after EAMG received MSCs, The expressions of CD28 on CD4+T cell in treatment group were decreased compared with the control group(p﹤0.05)and were no difference compared with the healthy group(p﹥0.05). The expressions of CD28 on CD4+T cell in control group were increased compared with the healthy group(p﹤0.05). The expressions of CTLA-4 on CD4+T cell in treatment group were increased compared with the control group and healthy group (p<0.01). The expressions of CTLA-4 on CD4+T cell in control group were increased compared with the healthy group (p﹤0.05). The proportion of CD4+CD25+regulatory T cell in treatment group were increased compared with the control group and healthy group (p<0.01).There were no difference between the control group and healthy group (p﹥0.05).Conclusions:1. Mesenchymal stem cells can be successfully isolate and cultivate from rat bone marrow and could be used in cell therapy.2. Immunization with the synthetic Rα97-116 peptide can induce EAMG in Lewis rats and provide a simple and available animal models for basic researches of MG.3. MSCs Transplantation could ameliorate muscular weakness in EAMG rats involved in decreased levels of anti-AChR antibodies and suppressed T cell activation,up-regulating CD25 and CTLA-4 expressing on CD4+T cell. |
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