| Objective:In this experiment,bone marrow mesenchymal stem cell was transplanted into the blood of experimental autoimmune uveitis rat,the clinical observation of the impact on uveitis rats was performed to analyze its effects on the immune mechanisms in vivo,with a view to providing a reliable theoretical basis and experimental foundation for the clinical treatment of uveitis.Methods:(1) culture and identification of bone marrow mesenchymal stem cells rat was executed by cervical spine dislocation to get femur under sterile conditions,the bone marrow was punched into the culture bottle with DMEM and inoculated into two culture bottle.The density of cell inoculated was 5×106/ml,Subculture every 3～4 d. When grow to about 90%of fusion,the cells were digested with 0·25%trypsin at room temperature,and inoculated at the ratio of 1:2.subculture repeatedly until the third generation.Applicate FACScan flow cytometry to detect surface antigens(CD45,CD71, CD44 and CD105) expression of mesenchymal stem cells.Finally made cell suspension with PBS,the number of the cells was 2×106/ml.(2) Grouping and Induction of EAU rats 18 Lewis rats were randomly divided into model control group,intervention group and normal control group,six in each group.Mix 1mg/ml HS-AgP35 solution and complete Freund's adjuvant and inject 0.2ml sufficient emulsified s-antigen in each hind foot pad,Superadditional the same dose S-antigen was given after seven days.(3) bone marrow mesenchymal stem cells transplantation when the emulsified s-antigen was first injected,lml bone marrow mesenchymal stem cell suspension was injected into tail vein of the intervention group rats.Animals were examined for two weeks time. (4) EAU clinical observation clinical observation was performed every day after immune.including the anterior segment and fundus examination,note a detailed record of clinical performance of three groups,with reference to the Caspi's clinical classification.(5) Histopathology observation Kill the animals at the 7th day after the second immune,extract eyeballs,make pathological section conventionally,examine the sample with light microscope and compare the Pathological changes among the three groups.(6) IFN-γdetection 2mL peripheral blood was taken from animals in each rat, and centrifugated.test IFN-γlevel in serum of each rat with ELISA.Results:①the third generation of bone marrow-derived mesenchymal stem cells were spindle and adherent After repeated subculture.Cell surface antigen expression tested by Flow cytometry:CD44(+),CD45(-),CD71(+) and CD 105(+)②The clinical grading of EAU rats in model control group was 1 rat at grade 1(16.67%),1 rat at grade2(16.67%),3 rats at grade 3(50%),1 ratatgrade4(16.67%);and 2 rats at grade 0(33.33%),3 rats at grade 0.5(50%),1 rat at grade 1(16.67%)in intervention group.Compared with model control group.The difference has statistical significance(P =0.028).③The histopathological grading of EAU rats in model control group was 4 rats at grade 3(66.67%),2 rats at grade 4(33.33%);and 1 rat at grade 0(16.67%),4 rats at grade 0.5(66.67%),1 rat at grade 1(16.67%) in intervention group.The difference between the two groups was statistical significance(P＜0.01).④IFN-γlevel in peripheral blood of intervention group rats declines,compared with model control group.the difference was statistical significance(P＜0.05).Conclusion:①Clinical manifestations and histopathological changes in EAU rats which injected with MSCs cell suspension were significantly lighter than the control group,shows that bone marrow-derived mesenchymal stem cells can reduce the severity of uveitis,and improve the condition of uveitis in rats;②INF-γlevels in MSCs injection group were significantly lower than the model control group,shows that bone marrow mesenchymal stem cells can inhibit sensitized T lymphocytes secreting INF-γ,and mitigate the damage to the organization.
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