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

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

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 Downregulation 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.