敲低CD2AP基因表达对足细胞增殖和分裂的影响

目的观察敲低CD2相关蛋白（CD2AP）基因表达对足细胞增殖和分裂的影响。方法采用RPMI 1640培养基,在33℃培养永生化小鼠足细胞系。以PKH-26红色荧光染料标记足细胞后,用Metafectene转染试剂转染针对CD2AP的小分子干扰RNA（siRNA）。转染24 h后以流式细胞仪检测转染效率;48 h后用RT-PCR和Western印迹检测转染后CD2AP mRNA和蛋白表达情况;72 h后以流式细胞仪检测足细胞增殖指数及细胞周期;用Oregon Green 488标记的Paclitaxel直接标记足细胞内的微管蛋白;用激光共聚焦显微镜检测双核及多核足细胞的比例。结果流式细胞仪结果显示,CD2AP特异性siRNA转染效率为66.27%。转染siRNA 48 h后,CD2AP mRNA和蛋白表达分别下降57%和39%。转染72 h后,足细胞增殖指数显著下降（P〈0.05）,G2/M期的细胞数量显著增多（P〈0.05）。共聚焦显微镜下可见转染CD2AP siRNA后,部分细胞有丝分裂后不能分离,双核及多核足细胞的比例显著增加（P〈0.05）。结论敲低CD2AP基因的表达能阻碍细胞有丝分裂后期的细胞分离,抑制足细胞的增殖能力。

Knocking-down CD2AP gene expression inhibits the proliferation and blocks the division of podocytes

Objective To study the effect of CD2-associated protein (CD2AP) on podocyte proliferation and division. Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃ permissive condition. After labeling with red fluorescent dye PKH-26, podocytes were transfected with CD2AP small interfering RNA (siRNA) using transfection reagent metafectene. Transfection efficiency was measured by flow cytometer 24 hours later and inhibitory effect of CD2AP siRNA was determined by RT-PCR and Western blot at 48 hours after transfection. Proliferation index and cell cycle of podocytes were examined by flow cytometer 72 hours later. Microbule of podocyte was detected by Oregon Green○Ｒ488-conjugated paclitaxel. The ratio of binuclear and polynuclear podocytes was counted under laser scanning confocal microscope (LSCM). Results The transfection efficiency of CD2AP siRNA was 66.27%. Forty-eight hours after transfection with specific siRNA, the expression levels of CD2AP mRNA and protein were down-regulated by 57％ and 39％ respectively. The podocyte proliferation index apparently descended (P<0.05) and the cells in the phase of G2/M accumulated significantly when detected at 72 hours (P<0.05). Under LSCM, podocytes that could not separate after M-period were easily found. The ratio of binuclear and polynuclear podocytes in CD2AP siRNA transfected group was markedly higher than that in control group (P<0.05). Conclusion Knocking-down CD2AP gene expression can interfer cell separation after mitosis and inhibit the podocyte proliferation.