解剖学报

解剖学报 ›› 2017, Vol. 48 ›› Issue (6): 669-674.doi: 10.16098/j.issn.0529-1356.2017.06.006

• 细胞和分子生物学 • 上一篇    下一篇

CD73 对骨髓间充质干细胞增殖与衰老的影响

王琰1 李琼1 张三林2 郭志坤1*   

  1. 1. 新乡医学院河南省医用组织再生重点实验室，河南 新乡 453003; 2. 中国人民解放军第371医院心血管内科，河南 新乡 453003
  • 收稿日期:2017-01-20 修回日期:2017-03-28 出版日期:2017-12-06 发布日期:2017-12-06
  • 通讯作者: 郭志坤 E-mail:gzk@xxmu.edu.cn
  • 基金资助:
    国家自然科学基金;河南省卫生厅攻关项目;新乡医学院研究生科研创新支持计划项目

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

WANG Yan1 LI Qiong1 ZHANG San-lin2 GUO Zhi-kun 1*   

  1. 1. He’nan Key Laboratory of Medical Tissue Regeneration, Xinxiang Medical University, He’ nan Xinxiang 453003, China; 2. Department of Cardiovascular Medicine, the 371st Hospital of Chinese PLA, He’nan Xinxiang 453003, China
  • Received:2017-01-20 Revised:2017-03-28 Online:2017-12-06 Published:2017-12-06
  • Contact: GUO Zhi-kun E-mail:gzk@xxmu.edu.cn

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摘要：

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）、CD73 RNAi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。

关键词:  CD73, 增殖, 衰老, 骨髓间充质干细胞, 超微结构, 流式细胞术, 小鼠

Abstract:

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-β-Gal) staining was used to detect the cell senescence. Cell proliferation was measured by MTT assay. Cell cycle was detected by flow cytometry. Ultrastructures 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.

Key words: CD73, Proliferation, Senescence, Bone marrow mesenchymal stem cell, Ultrastructure, Flow cytometry, Mouse