摘要目的 探讨S期激酶相关蛋白 2(Skp2)表达变化对系膜细胞增殖的影响.方法 设计合成大鼠Skp2 siRNA和对照siRNA、pIRES-GFP-Skp2质粒和pIRES-GFP质粒.采用脂质体转染法进行细胞转染.半定量PCR和Western印迹法检测Skp2的mRNA、蛋白表达.原代培养的大鼠系膜细胞以每孔3000个接种于96孔板,分为以下6组:(1)无血清组;(2)20%胎牛血清(FCS)组;(3)10%FCS+pIRES-GFP质粒转染组;(4)10%FCS+pIRES-GFP-Skp2质粒转染组;(5)20%FCS+对照siRNA组;(6)20%FCS+Skp2 siRNA组.四甲基偶氮唑盐(MTT)法检测细胞相对活力和相对细胞数;BrdU标记法检测S期细胞;流式细胞仪检测细胞周期.结果 半定量PCR法结果显示,与阴性对照siRNA组相比,Skp2 siRNA转染后Skp2 mRNA表达显著下调.Western印迹结果显示,与pIRES-GFP质粒转染组相比,pIRES-GFP-Skp2质粒转染后Skp2蛋白表达显著上调.MTT、BrdU和细胞周期分析显示,与相应对照组相比,Skp2质粒转染后相对细胞数增加(A值:0.419±0.088比0.305±0.036,P＜0.01)、S期细胞数增多(BrdU阳性细胞:0.21±0.04比0.15±0.03,P＜0.01;S期细胞数:20.18±0.64比14.33±0.37,P＜0.01);Skp2 siRNA转染后相对细胞数减少(A值:0.328±0.069比0.482±0.133,P＜0.01);S期细胞数减少(BrdU阳性细胞:0.17±0.01比0.24±0.00,P＜0.01;S期细胞数:16.52±0.75比23.81±1.25,P＜0.01).结论 Skp2过表达促进系膜细胞增殖,下调Skp2表达可抑制系膜细胞增殖.

abstractsObjective To explore whether the change of S phase kinase-associated protein 2 (Skp2) expression could regulate mesangial cell proliferation. Methods Skp2 siRNA and control siRNA, pIRES-GFP-Skp2 plasmid and pIRES-GFP plasmid were designed and synthesized. Cell transfection was performed by Lipofectamine 2000. Skp2 mRNA and protein levels were detected by semiquantitative PCR and Western blotting respectively. Primary culture rat mesangial cells were divided into 6 groups: 0%FCS, 20%FCS, 10%FCS+pIRES-GFP plasmid, 10%FCS+pIRES-GFP-Skp2 plasmid, 20%FCS+control siRNA, 20%FCS+Skp2 siRNA. Cell number was detected by MTT. S phase entry was measured by BrdU labeling. Cell cycle profile was determined by flow cytometric analysis. Results Skp2 mRNA level was significantly down-regulated by Skp2 siRNA compared to control siRNA. Skp2 protein level increased after pIRES-GFP-Skp2plasmid transfection compared to pIRES-GFP plasmid. MTT, BrdU labeling and cell cycle profile demonstrated that cell number (A: 0.419±0.088 vs 0.305±0.036, P＜0.01) and S-phase cells (BrdU labeling positive cell: 0.21±0.04 vs 0.15±0.03, P＜0.01;S-phase cell number:20.18±0.64vs 14.33±0.37, P＜0.01) obviously increased after Skp2 plasmid transfection, while decreased after Skp2 siRNA transfection compared to control groups (A: 0.328±0.069 vs 0.482±0.133, P＜0.01;BrdU labeling positive cell: 0.17±0.01 vs 0.24±0.00, P＜0.01; S-phase cell number: 16.52±0.75vs 23.81 ±1.25, P＜0.01). Conclusion Over-expression of Skp2 stimulates mesangial cell proliferation while down-regulated expression of Skp2 inhibits mesangial cell proliferation.