| Objective:1. To establish a method for isolation and culture of bone marrow mesenchymal stem cells(MSCs) from Lewis rats in vitro and to identify characteristics of the MSCs after culture expansion.2. To explore preparation of experimental autoimmune myasthenia gravis(EAMG) with rat 97-116(Rα97-116) peptide of the acetylcholine receptor(AChR)α-subunit and provide a simple and available animal model for the basis research of MG.3. To investigate the expression of transcription factor T-bet and GATA-3 in peripheral blood mononuclear cells (PBMCs) of EAMG and their functions in pathogenesis of EAMG..4. To study the effects of MSCs on the clinical manifestation and related immunity function of EAMG.Methods: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 prepare EAMG rat models in experiment group and only with PBS in control group. Clinical manifestation was evaluated by the measurement of body weight and Lennon clinical score. EAMG rats were further confirmed by 3,5 Hz repetitive nerve stimulation (RNS) for positive decremental respnse and ELISA assay for plasma AChR-Ab titers. A week after third immunization,20 EAMG rats were determined and divided randomly into MSCs transplantation group and model control group, each group had 10 rats,and 10 healthy rats with the same age were chosen as the normal control group. MSCs transplantation group were injected with 106 MSCs diluted in PBS (100μL) by caudal vein and an equal volume of PBS was injected in model control and normal control group.Then the body weight and Lennon clinical score of MSCs transplantation group and model control group were evaluated at the same time every day. The titers of anti-AChR in plasma were tested by ELISA after 2 weeks of the post-transplantation. The expression of T-bet,GATA-3 in PBMCs and IFN-γ,IL-4 in plasma were examinded by RT-PCR and ELISA respectively in three groups 3 weeks after transplantation.Results:1. 24 hours after primary culture , MSCs adhered to plastic surface of the culture dish.After 48～72 hours culture , the MSCs proliferated in colonies.These primary MSCs reached 70 %～80 % of confluence in 7～10 days, 97.5% of fifth generation of MSCs expressed CD90 and lacked expression (1.72% positive) of CD45.2. EAMG was confirmed in 70% of Lewis rats by the positive decremental response D5 >10% of 3,5 Hz RNS and the ratio >2.1 of the plasma AChR-Ab titers to control group.3. The 15th day after EAMG accepted MSCs, the body weight of rats in MSCs transplantation group was increased significantly compared with that of the model control group(p﹤0.05).4. The 13th day after EAMG accepted MSCs, the changes of the mean clinical score of rats in the cell transplantation group decreased (p﹤0.05).5. Compared with the model control group , the amount of anti-AChR IgG in serum in MSCs transplantation group decreased significantly (p﹤0.05) after 2 weeks of MSCs transplantation.6. 3 weeks after MSCs transplantation, the expression of T-bet mRNA was 0.67±0.13 in MSCs transplantation group and was 0.85±0.16 in the model control group, there was a significant difference between the two groups(p<0.01),and no significant difference was found between MSCs transplantation group and normal control group( p>0.05). The levels of GATA-3 mRNA expression was 0.51±0.11 in MSCs transplantation group and was 0.70±0.09 in the model control group, there was also a significant difference between the two groups(p<0.01),and no significant difference was found between MSCs transplantation group and normal control group( p>0.05). In MSCs transplantation group, plasma concentration of IFN-γwas 113.24±16.93 pg/ml,which was significantly lower than 231.71±44.44 pg/ml in the model control group (P<0.01), and no significant difference was found between MSCs transplantation group and normal control group(p>0.05).In MSCs transplantation group, plasma concentration of IL-4 was 87.33±3.50 pg/ml was significantly lower than 96.21±4.77 pg/ml in the model control group (p <0.01), no significant difference was found between MSCs transplantation group and normal control group( p>0.05). IFN-γand IL-4 were positively correlated respectively with T-bet and GATA-3 expression in model control group.(r=0.87, p<0.01;r=0.89, p <0.01 respectively).Conclusions:1. MSCs can be successfully isolated and cultivated from rat bone marrow and could be used in MSCs transplantation therapy.2. Immunization with the synthetic Rα97-116 peptide could induce EAMG in Lewis rats, which will provide a convenient and feasible animal model for basis research of MG..3. EAMG is associated with an up-regulation of T-bet and GATA-3 expression, which might be responsible for the enhancement of immunologic response in EAMG. 4. MSCs transplantation could effectively ameliorate muscular weakness and decrease AChR-Ab titers in EAMG rats , which might be a potential and feasible intervention way of MG.. The mechanism of therapy action in MSCs transplantation might be related to the decrease of T-bet/GATA-3 expression,which further influence both dysdifferentiation of Th1 and Th2 cells and cytokines secreted by them so as to downregulate AChR-specific B-cell responses and AChR-reactive T-cell function. |
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