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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 proliferation and senescence of bone marrow mesenchymal stem cells (BMSCs). Methods The BMSCs were cultured and the lentivirus CD73 overexpression vector and CD73 RNA interference vector were constructed and transfected into BMSCs respectively. The experimental groups were divided into three groups: the overexpression group (OE group), the CD73RNAi interference group (RNAi group) and the control group (CON group) . Aging-related β-galactosidase (SA-β-Gal) staining was used to detect cell senescence; Cell proliferation was measured by MTT assay; Cell cycle was detected by flow cytometry; Transmission electron microscopy Structure; non-invasive cell function analyzer to detect cell growth curve. Results The number of senescent cells RNAi group> CON group> OE group (P <0.05) at 72 h and 168 h after transfection of BMSCs. The proliferation ability of BMSCs was higher in OE group than in RNAi group and CON group (P <0.05). The changes of cell cycle in each group after 3 days of transfection were as follows: the proportion of G1 phase cells in OE group, RNAi group and CON group were 25.9%, 44.1% and 51.7%, respectively; Accounting for 48.8%, 30.9% and 26.9% respectively; the proportion of G2 phase cells was 25.2%, 25.0% and 21.4% respectively. Transmission electron microscopy showed that the content of endoplasmic reticulum and ribosome Increased; cell growth curve showed that the OE group was higher than the CON group and the RNAi group. Conclusion CD73 promotes the proliferation of BMSCs and inhibits the senescence of BMSCs.