骨髓间充质干细胞分离培养方法及其对CD8＋T淋巴细胞的免疫调控

Isolation and culture of bone marrow mesenchymal stem cells and their effects on immune regulation of CD8^+T lymphocytes

背景:骨髓间充质干细胞具有免疫调节特性,使其在免疫抑制方面有可预期的应用价值。目的:探讨骨髓间充质干细胞的分离培养方法及其对CD8^+T淋巴细胞的免疫调控。方法:采用骨髓直接贴壁法获得原代Wistar大鼠骨髓间充质干细胞,利用差速贴壁结合消化法纯化原代细胞,加入Ⅰ型胶原蛋白作为细胞外基质,扩增骨髓间充质干细胞。取第2代骨髓间充质干细胞,分别将0,1×10~3,1×10~4,1×10~5/well的骨髓间充质干细胞与CD8^+T淋巴细胞共培养,植物血凝素刺激68 h,荧光CFSE法观察CD8^+T淋巴细胞聚集,MTT法检测CD8^+T淋巴细胞增殖率。结果与结论:(1)不同密度骨髓间充质干细胞对T淋巴细胞均有抑制作用,1×10~3/well组抑制能力最弱,形成集落现象;1×105/well组抑制能力最强,细胞未见明显聚集现象;(2)1×10~3,1×10~4,1×10~5/well组CD8^+T淋巴细胞增殖率分别为(44.83±4.92)%、(31.94±6.28)%及(15.77±3.98)%,3组间两两比较差异均有显著性意义(P<0.05);(3)结果表明,采用全骨髓直接贴壁法能获得原代大鼠骨髓间充质干细胞,采用差速贴壁联合消化控制法能完成细胞纯化,骨髓间充质干细胞浓度依赖性抑制T淋巴细胞增殖。

BACKGROUND： Bone marrow mesenchymal stem cells have immunomodulatory properties and have potential applications in immunosuppression.OBJECTIVE： To investigate the isolation and culture of bone marrow mesenchymal stem cells and the immunoregulation of CD8＋T lymphocytes. METHODS： Primary bone marrow mesenchymal stem cells were isolated from Wistar rats by bone marrow adherence method. The primary cells were purified by differential adherence and digestion, and collagen type I was added as extracellular matrix to expand bone marrow mesenchymal stem cells. Passage 2 bone marrow mesenchymal stem cells at densities of 0, 1×10^3, 1×10^4 and 1×10^5/well were co-cultured with CD8＋T lymphocytes, followed by phytohemagglutinin stimulation for 68 hours. T lymphocyte aggregation and proliferation were detected by staining with staining with 5,6-carboxyfluorescein diacetate succinimidyl ester and MTT, respectively. RESULTS AND CONCLUSION： Different concentrations of bone marrow mesenchymal stem cells had an inhibitory effect on T lymphocytes, but this effect was weakest for the cells at the density of 1×10^3 per well and strongest for the cells at the density of 1×10^5 per well. Obvious agglomeration was observed in the same media. The proliferation rates of CD8＋T lymphocytes were（44.83±4.92）%,（31.94±6.28）% and（15.77±3.98）%, respectively, after co-culture with 1×10^3, 1×10^4, 1×10^5/well bone marrow mesenchymal stem cells. There were significant differences between groups（P〈0.05）. These results showed that primary rat bone marrow mesenchymal stem cells could be obtained by direct adherence method of the whole bone marrow, and the cells could be purified by differential adherence combined with digestion and control method. Bone marrow mesenchymal stem cells could inhibit T lymphocyte proliferation in a concentrationdependent manner.