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中华关节外科杂志(电子版) ›› 2019, Vol. 13 ›› Issue (01) : 52 -57,51. doi: 10.3877/cma.j.issn.1674-134X.2019.01.011

所属专题: 文献

基础论著

微小RNA-564基因下调对滑膜间充质干细胞成软骨分化的影响
孙祥燚1, 张雷1, 陈烁1, 周利武1,(), 赵建宁1   
  1. 1. 210002 南京大学医学院附属金陵医院(东部战区总医院)骨科
  • 收稿日期:2018-07-24 出版日期:2019-02-01
  • 通信作者: 周利武
  • 基金资助:
    国家自然科学基金青年科学基金项目(81702170)

Effects of microRNA 564 gene expression down-regulation on chondrogenic differentiation of synovial mesenchymal stem cells

Xiangyi Sun1, Lei Zhang1, Shuo Chen1, Liwu Zhou1,(), Jianning Zhao1   

  1. 1. Department of Orthopedics, Jinling Clinical Medical College (Eastern Theater General Hospital), Nanjing University, Nanjing 210002, China
  • Received:2018-07-24 Published:2019-02-01
  • Corresponding author: Liwu Zhou
  • About author:
    Corresponding author: Zhou Liwu, Email:
引用本文:

孙祥燚, 张雷, 陈烁, 周利武, 赵建宁. 微小RNA-564基因下调对滑膜间充质干细胞成软骨分化的影响[J]. 中华关节外科杂志(电子版), 2019, 13(01): 52-57,51.

Xiangyi Sun, Lei Zhang, Shuo Chen, Liwu Zhou, Jianning Zhao. Effects of microRNA 564 gene expression down-regulation on chondrogenic differentiation of synovial mesenchymal stem cells[J]. Chinese Journal of Joint Surgery(Electronic Edition), 2019, 13(01): 52-57,51.

目的

探究下调微小RNA-564(miR-564)基因对滑膜间充质干细胞成软骨分化的作用。

方法

取第3代的人滑膜间充质干细胞(SMSCs)作为实验细胞,实验设计为3组:SMSCs空白对照组(空白组);miRNA抑制剂转染SMSCs对照组(对照组);miR-564抑制剂转染SMSCs实验组(实验组)。将3组SMSCs同时成软骨诱导培养,观察诱导后软骨细胞的组织形态,并检测诱导前7 d内的3组细胞增殖曲线,和软骨分化特异性基因和蛋白[Ⅱ型胶原、蛋白聚糖、性别决定区Y框蛋白9(Sox9)、母亲DPP同源物4(Smad4)];本次实验所有数据均采用单因素方差分析(one-way ANOVA)检验。

结果

甲苯胺蓝染色观察细胞形态与特点基本符合软骨细胞,实验组细胞数量更多,蓝染更明显;细胞增殖曲线表明实验组细胞增殖速度明显快于对照组(F=0.842, P <0.01);RT-PCR检测与Western Blotting检测表明实验组软骨细胞分化特异基因和蛋白表达较对照组明显升高(F=2274.75, F=447.31, F=30476.22; P <0.01),并且实验组TGF-BMP关键基因及蛋白Smad 4有所升高(F=457.02, P <0.01)。

结论

下调miR-564基因的表达可促进滑膜间充质干细胞向软骨细胞增殖与分化,并且有可能是通过作用于TGF-BMP通路实现。

Objective

To investigate the effect of down-regulation of microRNA-564(miR-564) gene on chondrogenic differentiation of synovial mesenchymal stem cells.

Methods

The third passage of SMSCs was used for the experiment. The experimental design was as follows: the blank group of SMSCs; the control group of miRNA inhibitor transfected with SMSCs; the experimental group of miR-564 inhibitor transfected into SMSCs. The three groups were cultured and induced for chondrogenesis. The histomorphology of chondrocytes was observed and the three cell proliferation curves were detected within 7 d before induction. Finally, the cartilage differentiation-specific genes and proteins in the three groups of SMSCs were detected, including collagen, proteoglycan, sex related Y protein-box 9(Sox 9), SMAD family member 4(Smad4). All the data were analyzed by one-way ANOVA test.

Results

The morphology and characteristics of the cells were consistent with chondrocytes. The experimental group showed more cells and the toluidine blue staining of the experimental group was more obvious. The proliferation rate of the experimental group was significantly higer than that of the control group (F=0.842, P<0.01). Reverse transcription-PCR(RT-PCR) and western blot showed that the specific genes and protein expression of chondrocyte differentiation in the experimental group were significantly higher than those in the control group (F=2 274.75, F=447.31, F=30 476.22; all P <0.01), and the key genes of TGF-BMP signaling pathway(TGF-BMP)and Smad 4 protein increased significantly in the experimental group(F=457.02, P <0.01).

Conclusion

Down-regulation of miR-564 gene expression could promote the proliferation and differentiation of synovial mesenchymal stem cells into chondrocytes, and may be achieved by the action of TGF-BMP pathway.

表1 各基因引物系列
图1 SMSCs形态学观察及转染效果检测。图A示培养2 d时SMSCs形态(×100);图B示培养4 d时SMSCs形态(×100);图C为SMSCs细胞表明抗原鉴定:CD29、CD73呈阳性表达,CD14、CD68呈阴性表达;图D为3组SMSCs转染后,miR-564基因的表达情况(F =2 042.93,*P <0.01)
图3 SMSCs(滑膜间充质干细胞)成软骨诱导21 d后的甲苯胺蓝染色(×100)。图A为空白对照组;图B为miRNA抑制剂对照组;图C为实验组,可见实验组蓝染细胞多于其他两组
图4 软骨分化基因检测比较。图A示实验组的软骨特异性基因表达在强于空白和对照组(F =2 274.75,F=447.31,F=3 0476.22;*P <0.01);图B示TGF-BMP(转化生长因子-骨形态发生蛋白)通路关键基因Smad 4的表达实验组高于其他两组(F =457.02,*P <0.01)
图5 诱导后3组SMSCs(滑膜间充质干细胞)软骨相关蛋白表达的western blot电泳图,示实验组各软骨相关蛋白表达均高于其他两组
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