稳定过表达饰胶蛋白聚糖基因对大鼠肾系膜细胞凋亡的影响

Pro-apoptotic effect of decorin on rat mesangial cells in vitro

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。

Objective To investigate the effects of over-expression of decorin （DCN） gene on apoptosis of cultured rat mesangial cells （MsC）. Methods PcDNA3.1A-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 apoptotic 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.