摘要目的 观察稳定过表达饰胶蛋白聚糖(DCN)基因对大鼠肾系膜细胞(MsC)凋亡的影响.方法 采用脂质体介导法将pcDNA3.1A-DCN质粒稳定转染MsC,G418筛选阳性克隆.细胞免疫荧光法、逆转录-聚合酶链反应(RT-PCR)和Western印迹法鉴定并获得转染DCN阳性的细胞株(MsC/DCN);脂质体介导法将DCN-siRNA瞬时转染MsC/DCN以干扰DCN表达,Western印迹法鉴定.Hoechst染色和流式细胞仪观察细胞凋亡及比率,Western印迹法检测活性凋亡相关激酶半胱氨酸蛋白酶3(Caspase-3)蛋白的表达.结果 成功建立了过表达DCN基因的细胞株MsC/DCN.MsC/DCN中凋亡率为(20.40±8.01)%,显著高于MsC的凋亡率(2.07±0.99)%(P＜0.01),部分MsC/DCN发生典型的形态学改变.活性Caspase-3的蛋白表达也明显高于MsC(P＜0.01).DCN-siRNA瞬时转染不仅有效干扰了MsC/DCN中DCN的表达和降低了细胞凋亡率,而且显著下调了活性Caspase-3的蛋白表达.结论 大鼠MsC中DCN基因的过表达能诱导MsC发生凋亡,为肾小球疾病中调控MsC增生提供了新的实验依据.

abstractsObjective To investigate the effects of over-expression of decorin (DCN) gene on apoptosis of cultured rat mesangial cells (MsC).Methods PcDNA3.1 A-DCN plasmid was transfected into cultured rat MsC by the induction of liposome and positive clones were selected by treating the cells with G418.The MsC clones stably expressing DCN (MsC/DCN) were confirmed by cellular immunofluorescence,reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot.The DCN-siRNA was used for blocking DCN expression in MsC/DCN,and was confirmed by Western blot.The apoptosis of MsC was assayed by flow cytometry and Hoechst staining.Expression of Caspase-3 was assayed by Western blot.Results Positive clones with DCN over-expression were established.The apoptotic rate in MsC/DCN was(20.40±8.01)% and was much higher than the (2.07±0.99)% in MsC (P＜0.01).Some of the MsC/DCN cells showed typical morphologic changes of apoptosis.The protein expression of active Caspase-3 was also significantly increased in MsC/DCN compared to MsC(P＜0.01).DCN-siRNA transfection not only significantly blocked the expression of DCN and reduced the rate of apoptotie cells,but also down-regulated the expression of active Caspase-3.Conclusions Over-expression of DCN induces apoptosis of cultured rat MsC in vitro.This effect of DCN inducing apoptosis suggests a novel strategy for regulating the proliferation of MsC in glomerular diseases.