CD73对骨髓间充质干细胞增殖与衰老的影响

Effect of CD73 on the proliferation and senescence of bone marrow mesenchymal stem cells

目的探讨CD73对骨髓间充质干细胞(BMSCs)增殖与衰老的影响。方法培养小鼠BMSCs,构建慢病毒CD73过表达载体和CD73 RNAi干扰载体,分别转染BMSCs,实验分组:CD73过表达组(OE组)、CD73RNAi干扰组(RNAi组)和对照组(CON组)。衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测细胞的衰老情况;MTT法检测细胞增殖能力变化;流式细胞术检测细胞周期变化;透射电子显微镜观测转染后细胞的超微结构;非损伤细胞功能分析仪检测细胞生长曲线。结果慢病毒转染BMSCs后72 h和168 h,衰老细胞数量RNAi组>CON组>OE组(P<0.05);转染后3~4d BMSCs的增殖能力为OE组较RNAi组和CON组细胞增殖速度快(P<0.05);转染后3d各组细胞周期的变化是:OE组、RNAi组和CON组的G1期细胞所占比例分别为25.9%、44.1%和51.7%;S期细胞所占比例分别为48.8%、30.9%和26.9%;G2期细胞所占比例分别为25.2%、25.0%和21.4%;透射电子显微镜观察结果显示,OE组细胞质内粗面内质网和核糖体增多;细胞生长曲线显示OE组高于CON组和RNAi组。结论 CD73促进BMSCs增殖且抑制BMSCs衰老。

Objective To investigate the effect of CD73 on the proliferation and senescence of bone marrow mesenchymal stem cells （ BMSCs）. Methods The mouse BMSCs were cultured in vitro. We constructed lentivirus of CD73 overexpression and CD73 RNAi vectors and transfected into the BMSCs. The experimental groups were divided into three groups： CD73 overexpression group （OE group）, CD73 RNAi interference group （RNAi group） and control group （ CON group）. Galactosidase （ SA-13-Gal） staining was used to detect the cell senescence. Cell proliferation was measured by MTT assay. Cell cycle was detected by flow cytometry. Uhrastructures were observed by transmission electron microscopy （TEM）. The cell growth curve was detected by non-injured cell function analyzer. Results The number of senescent cells in RNAi group was significantly higher than that in CON and OE groups （ P 〈 0. 05 ） at 72 hours post-transfection. The proliferation of BMSCs in OE group was significantly higher than CON and RNAi groups at 72 and 96 hours post-transfection （P 〈 0.05 ）. The percentages of G1 phase cells in OE group, RNAi group and CON group were 25.9% , 44. 1% and 51.7% , respectively, and that of S phase cells were 48.8% , 30.9% and 26.9% , respectively. The percentage of cells in G2 phase were 25.2% , 25.0% and 21.4% , respectively. The result of transmission electron microscopy showed that the cell rough endoplasmic reticulum and the ribosomes were increased in the OE group. The cell growth curve showed that the cell proliferation ability in OE group was higher than that in CON group and RNAi group. Conclusion CD73 promotes BMSCs proliferation and inhibits BMSC senescence.