NG2蛋白多糖促进肾小球系膜细胞增殖和细胞外基质积聚

目的 探讨NG2蛋白多糖参与肾小球硬化过程的可能机制。 方法 构建针对NG2的特异性shRNA（Psilencer-NG2）和对照shRNA（Psilencer-NC）。将Psilencer-NG2、Psilencer-NC和全长型NG2真核表达载体pcDNA/NG2以及空质粒pcDNAⅠ分别转染到体外培养的大鼠系膜细胞（RMC）。实时定量PCR和Western印迹检测转染质粒对NG2 的干扰效率和过表达效率；MTT法观察各组细胞增殖情况；流式细胞仪检测细胞周期变化；实时定量PCR检测各组层粘连蛋白（laminin）的表达。 结果 转染pcDNA/NG2后的HBZY-1细胞NG2 mRNA和蛋白表达水平均明显增加（P < 0.05，P < 0.05），而针对NG2的特异性shRNA干扰导致HBZY-1细胞NG2 mRNA和蛋白表达水平均显著下调（P < 0.01；P < 0.01），分别表明达到过表达和沉默NG2的效应。过表达NG2后，RMC中laminin β1表达显著增加（1.25±0.04比 1.00±0.09，P < 0.05）；RMC增殖明显，处于S期的细胞数明显增加，而G0/G1期的细胞数减少。而沉默NG2后，laminin β1表达显著减少（0.81±0.02比1.00±0.08，P < 0.05）；RMC增殖缓慢，处于G0/G1期的细胞数明显增加，而S期的细胞数减少。 结论 NG2蛋白多糖可能通过促进系膜细胞增殖和细胞外基质积聚参与肾小球硬化的过程。

NG2 proteoglycan promotes mesangial cells proliferation and extracellular matrix production

Objective To explore the role of NG2 proteoglycan in the pathogenesis of glomerulosclerosis. Methods Eukaryotic expression vectors carrying the small hairpin RNA (shRNA) for NG2 mRNA , named as Psilencer-NG2, was constructed. Then, rat mesangial cells (RMC) were transfeeted with Psilencer-NG2, Psilencer-NC (negative control), pcDNA/NG2 (NG2 over-expressive vector) and empty vector pcDNA 1 respectively. The expression of endogenous NG2 in RMCs was examined by real-time PCR and Western blotting. Cell proliferation was analyzed by MTT assay and flow cytometry. The expression of laminin was detected by real-time PCR. Results Transfection of pcDNA/NG2 into HBZY-1 cells resulted in over-expression of NG2 mRNA and protein (P<0.05, P<0.05). Transfection of Psilencer-NG2 led to reduced expression of NG2 mRNA and protein (P<0.01, P<0.01). The expression of laminin β1 significantly increased due to overexpression of NG2 and decreased by treating with NG2 siRNA. According to MTT assay, overexpreasion of NG2 significantly stimulated the proliferation of mesangial cells while NG2 silencing inhibited it. NG2 increased the cell number in S phase and decreased the cell number in G0/G1 phase, while silencing NG2 induced the decrease of cell number in S phase and the increase of cell number in G0/G1 phase. Conclusion NG2 actively participates in the pathogenesis of glomerulosclerosis by stimulating proliferation of RMCs and increasing the deposition of ECM.