Klf4抑制Wnt/β-catenin信号通路调控软骨细胞增殖的研究

Study on the regulation of chondrocyte proliferation by Klf4 inhibiting Wnt/β-catenin signaling pathway

目的探讨Klf4基因对大鼠软骨细胞增殖影响及相关机制。方法从出生24 h SD幼鼠的股骨头提取软骨细胞,细胞免疫荧光鉴定Col2a1和Sox9基因的表达。转染慢病毒载体PCDH-GFP-Klf4和辅助质粒PSPAX2及PMD2.0G至293ft细胞中产生慢病毒,48 h后收集病毒上清,用其感染大鼠软骨细胞作为Klf4组,用PCDH-GFP慢病毒感染细胞作为对照组。采用CCK8法检测2组细胞生长增殖变化;采用Western blot方法,分别检测2组细胞中Klf4基因表达、增殖相关基因PCNA和CyclinD1蛋白表达、Wnt/β-catenin信号通路上下游基因表达情况。结果原代培养软骨细胞均强表达Col2a1和Sox9基因。慢病毒PCDH-GFP-Klf4和PCDH-GFP成功感染软骨细胞,Klf4基因在软骨细胞过表达。Klf4组软骨细胞生长明显受到抑制,PCNA和CyclinD1蛋白表达和Wnt/β-catenin信号通路相关基因表达均明显下调。结论过表达Klf4基因可能通过抑制Wnt/β-catenin信号通路相关分子的表达而抑制软骨细胞增殖。

Objective It is to investigate the effect of Klf4 gene on the proliferation of rat chondrocytes and its related mechanisms.Methods The chondrocytes were extracted from the femoral head of 24 hours old SD rats,and the expression of Col2a1 and Sox9 genes were identified by immunofluorescence.Lentivirus was generated by transfecting the lentiviral vector PCDH-GFP-Klf4 and the helper plasmids PSPAX2 and PMD2.0G to 293 ft cells.After 48 h,the virus supernatant was collected to infect the rat chondrocytes which used as Klf4 group,the cells infected with PCDH-GFP lentivirus were used as a control group.The growth and proliferation of the two groups were detected by CCK8 method.The expression of Klf4 gene,proliferation related gene PCNA and CyclinD1 protein and the expression of upstream and downstream genes of Wnt/β-catenin signaling pathway in the two groups were detected by Western blot.Results Primary cultured chondrocytes strongly expressed Col2a1 and Sox9 genes.Lentivirus PCDH-GFP-Klf4 and PCDH-GFP successfully infected chondrocytes,and Klf4 gene was overexpressed in chondrocytes.The growth of chondrocytes in Klf4 group was significantly inhibited,and the expression of PCNA and CyclinD1 protein and related protein of Wnt/β-catenin signaling pathway were down-regulated.Conclusion Overexpression of Klf4 may inhibit chondrocyte proliferation by inhibiting the expression of Wnt/β-catenin signaling pathway-related molecules.