骨髓间充质干细胞体外对1型糖尿病大鼠淋巴细胞的免疫调节作用

The Immunoregulation effects of bone marrow mesenchymal stem cells on the lymphocytes of rats with type 1 diabetes mellitus in vitro

目的 观察骨髓间充质干细胞（MSCs）体外对1型糖尿病（T1DM）大鼠淋巴细胞表型及增殖能力的影响,探讨其抑制淋巴细胞增殖的机制.方法 分离、培养和鉴定大鼠MSCs,噻唑蓝（MIT）比色法观察该细胞对淋巴细胞增殖能力的影响,应用流式细胞术分析MSCs对植物血凝素（PHA）作用下淋巴细胞凋亡,周期水平和CD4＋CD25＋调节性T细胞亚群（CD4＋CD25＋Tregs）比例的影响.结果 大鼠MSCs表型为CD29＋、CD90＋、CD106＋、CD34-、CD45-,对PHA刺激的淋巴细胞增殖有抑制作用,以淋巴细胞：MSCs为1∶1时（C组）抑制作用最强;共培养体系中,大部分淋巴细胞处于G0/G1期;C组淋巴细胞凋亡水平（58.05±0.89）%显著高于对照组（43.35±0.86）%（P＜0.05）;CD4＋CD25＋Tregs的比例C组（22.76±1.15）%显著高于对照组（5.80±0.68）%（P＜0.05）.结论 MSCs体外可显著抑制PHA刺激的T1DM大鼠淋巴细胞的增殖,其机制与CD4＋CD25＋Tregs比例增高密切相关.

Objective To observe the effects of bone marrow mesenchymal stem cells （MSCs） on the lymphocytes of rats with type 1 diabetes mellitus （T1DM） in vitro, and investigate the inhibitory effect of MSCs on lymphocytes proliferation and the underlying mechanism. Methods MSCs were isolated from SD rats, cultured in vitro, purified and then identified by testing the phenotypes with flow cytometry （FCM）. The third-generation MSCs were planted in 24-well plates. After treated with mitomycin C, MSCs were co-cultured for 72 h with the T1 DM rat＇s lymphocytes activated by phytohemagglutinin （PHA）. The proliferation of lymphocyte was measured by methyl thiazol tetrazolium （MTT） method. FCM analysis was done to investigate the apoptosis, cell cycle and the proportion of CD4＋ CD25＋ regulatory T cells of the T1 DM rat＇s lymphocytes after co-cultivation. Results The phenotypes of MSCs from normal SD rats were CD29 ＋ , CD90 ＋, CD106 ＋ , CD34-, CD45 -. MSCs obviously inhibited the lymphocyte proliferation stimco-culture system, most of the lymphocytes were arrested at G0/G1 phase. The apoptosis rate of lymphocytes （58.05 ± 0. 89）% in group C was increased significantly as compared with the control group （43.35± 0.86 ） % （ P 〈 0. 05 ） as well as the proportion of CD4 ＋ CD25 ＋ regulatory T cells （22.76 ± 1.15 ） % vs （5.80 ± 0. 68） %. Conclusion In vitro, MSCs can obviously inhibit the T1 DM rat＇ s lymphocytes proliferation stimulated with PHA via increasing the proportion of CD4 ＋ CD25 ＋ regulatory T cells.