MRL/lpr鼠骨髓间充质干细胞对T淋巴细胞作用的研究

Immunoregulatory effects of bone marrow-derived mesenchymal stem cells from MRL/Ipr mice on T lymphocytes

目的探讨狼疮鼠(MRL/lpr)及正常鼠(C57BL/6)骨髓间充质干细胞(MSCs)体外生物学特性、对T淋巴细胞作用的影响。方法采用直接贴壁筛选法分离培养扩增两种鼠系骨髓MSCs,流式细胞术鉴定MSCs表面标记物。观察MSCs生长状况并绘制生长曲线;取C57BL/6鼠脾脏细胞,经尼龙毛柱分选CD3^+T淋巴细胞,经刀豆蛋白A(ConA)刺激,取正常及狼疮鼠MSCs或MSCs培养上清与T细胞共培养72h,然后羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)染色测定T细胞增殖,流式细胞术检测共培养前后T细胞活化及T细胞的凋亡率。结果第2代(P2)后狼疮鼠MSCs生长增殖快于正常鼠MSCs;正常及狼疮鼠MSCs共培养均可抑制正常鼠T细胞的增殖(P<0.05),与MSCs上清共培养对正常鼠T细胞增殖无抑制作用(P>0.05);与两种MSCs或上清共培养均可降低正常T细胞的活化(P<0.05)及凋亡率(P<0.05)。对正常T细胞增殖、活化、凋亡的影响,狼疮鼠MSCs与正常鼠MSCs间差异无统计学意义(P>0.05)。结论狼疮鼠MSCs在体外生长上存在异常,但对T细胞增殖、活化、凋亡免疫调节无异常。

Objective To explore the biological characteristics and to compare the different regulation mechanisms of bone marrow mesenchymal stem cells （MSCs） derived from both lupus （MRL/lpr） and normal （C57BL/6） mice on T lymphocytes in vitro. Methods MSCs from MRL/lpr and C57BL/6 mice bone marrow were isolated and cultured. The surface phenotypes were detected by flow cytometry （FCM）. The morphological changes of MSCs were evaluated in primary and passage cultures. The growth curves were assayed. CD3^＋ T lymphocytes from spleen of C57BL/6 mice were isolated by nylon wool columns and stimulated by ConA and co-cultured with or without the two strains of MSCs or supernatant for 72 h. The proliferation of T cells stained by CFSE and activation of T cells were detected. The apoptosis was assessed by flow cytometry using Annexin V/PI. Results The growth of lupus MSCs was faster than normal controls （P〈0.05）. Both groups were positive for CD106 and CD105, and negative for CD34 and CD45. Both of the two MSCs could inhibit the proliferation of T cells whereas supernatant could not. The expression of CD69 and CD25 on CD3^＋ T cells stimulated by ConA was lower when co-cultured with two MSCs or supernatant than that in control group （P〈0.05）. A decrease of T cell apoptosis was seen when MSCs or supernatant of MRL/lpr or C57BL/6 were added （P〈 0.05）. No difference between the two groups. Conclusion MSCs from lupus mice present abnormal proliferation pattern in vitro. There is no evident defection in some immunoregulatory functions of lupus MSCs on T cells, such as proliferation, activation, apoptosis, compared with normal controls.