Ki67法检测间充质干细胞对淋巴细胞增殖抑制能力

Inhibitory effect of mesenchymal stem cells on lymphocyte proliferation was detected by Ki67 assay

目的 对比Ki67法与羧基荧光素乙酰乙酸(carboxyfluoreseein di-acetate succinimidyl ester,CFSE)法检测间充质干细胞(mesenchymal stem cell,MSCs)对淋巴细胞的增殖抑制能力,开发MSCs免疫调控功能评价的新方法。方法 选取5批脐带来源的MSCs,流式细胞术进行表面标志物(阳性CD73/CD90/CD105/CD44、阴性CD34/CD45/CD14/CD19)检测;试剂盒诱导后染色进行成脂、成骨、成软骨三系分化能力检测;流式细胞术进行增殖指数检测;CCK8法进行群体倍增时间检测;生物学有效性检测合格后与相同供者来源的外周血单个核细胞(peripheral blood mononuclear cell,PBMC)进行共培养,分别用CFSE法与Ki67法检测MSCs对PBMC的增殖抑制率。结果 制备的MSCs生长状态良好,CD73/CD90/CD105/CD44表达率均≥98%,CD34/CD45/CD14/CD19表达率均≤3%,成脂、成骨、成软骨三系分化能力良好,增殖指数为35.8±9.9,倍增时间为(19.1±1.2) h,增殖情况良好。CFSE法检测5批MSCs对PBMC的增殖抑制率为(91.20±0.70)%,Ki67法检测5批MSCs对PBMC的增殖抑制率为(89.99±1.45)%,2种方法比较,差异无统计学意义。结论 Ki67法可作为评价MSCs免疫调控功能的新方法,有效避免传统方法在检测过程中的局限性。

Objective Ki67 method was compared with carboxyfluoreseein di-acetate succinimidyl ester(CFSE) method was used to detect the inhibition of mesenchymal stem cells(MSCs) on lymphocyte proliferation, to proposed a new idea for the evaluation of the immunoregulatory function of MSCs. Methods Five batches of MSCs from umbilical cord were selected. Surface markers(positive CD73/CD90/CD105/CD44, negative CD34/CD45/CD14/CD19) were detected by flow cytometry. After induction of the kit, staining was performed to detect the differentiation ability of lipid, osteogenic and chondrogenic lines. Proliferation index was detected by flow cytometry. CCK8 method was used to detect population doubling time. After qualified biological validity test, the Peripheral blood mononuclear cells(PBMC) from the same donor were co-cultured with MSCs,and then, CFSE and Ki67 methods were used to detect the inhibition rate of MSCs on lymphocyte proliferation, respectively. Results The expression rates of CD73/CD90/CD105/CD44 were ≥ 98%, and CD34/CD45/CD14/CD19 were ≤ 2%. The proliferation index of MSCs was 35.8±9.9, and the multiplication time was(19.1±1.2) h. The proliferation was good. The proliferation inhibition rate of five batches of MSCs on PBMC was(91.20±0.70) % by CFSE method, and(89.99±1.45)% by Ki67 method. There was no statistical difference between the two methods. Conclusion Ki67 method can be used as a new method to evaluate the immunoregulatory function of MSCs, so as to avoid the limitations of traditional methods in the detection process.