NG2基因小发夹结构RNA抑制高糖诱导的大鼠系膜细胞增殖

Small hairpin RNA targeting NG2 gene inhibits the high glucose-induced proliferation of rat mesangial cells

目的构建针对NG2基因的小发夹结构RNA（shRNA）真核表达载体，观察该shRNA诱导的RNA干扰（RNAi）对高糖环境下大鼠肾小球系膜细胞（RMC）增殖的影响。方法体外培养RMC，高糖（30mmol／L）刺激24h，观察NG2基因表达的变化。设计两条针对NG2mRNA不同靶序列的干扰序列，构建针对NG2基因的shRNA表达载体Pgenesil—siNG21和Pgenesil-siNG22；选择不与任何基因同源的序列NC作为阴性对照。运用脂质体将3种质粒分别转染RMC，24h后荧光倒置显微镜观察转染效率，即增强绿色荧光蛋白（EGFP）表达率，荧光定量PCR法和Western印迹法观察NG2的干扰效率。以高糖刺激转染的RMC，用四甲基偶氮唑蓝比色法（MTY）和流式细胞仪（FCM）检测RMC增殖的变化。结果与低糖和甘露醇对照组比较，高糖刺激RMC后NG2表达水平明显升高，分别增高（65±32）％和（63±28）％，差异有统计学意义（P〈0．01）。质粒转染RMC24h后，EGFP的表达率为（50±10）％。高糖刺激后，干扰质粒转染组RMC增殖缓慢，与对照组比较差异有统计学意义（P〈0．01）。结论针对NG2基因的shRNA对高糖诱导的RMC增殖有明显的抑制作用。

Objective To construct eukaryotic expression vectors carrying the small hairpin RNA （shRNA） for NG2 mRNA and to study the effect on proliferation of rat mesangial cells （RMC） induced by high glucose stimulation through transfection and expression of shRNA targeting NG2 gene. Methods RMC was cultured in vitro and treated with high glucose （30 mmol/L） for 24 h. The change of NG2 expression was observed. Oligonucleotide hairpin sequences targeting NG2 gene were designed by the internet tool and was then inserted into the plasmid Pgenesil-1 containing enhanced green fluorescence protein （EGFP） to form plasmid Pgenesil-siNG21 and Pgenesil-siNG22. Plasmid expressing irrelevant shRNA was used as negative control named Pgenesil-siNC. RMC was transfected with above three kinds of plasmids respectively. Then the cells were stimulated with high glucose, and the changes of cell proliferation were observed by MTY and flow cytometry （FCM） methods. Results The expression of NG2 mRNA in RMC was significantly increased （P〈0.01） after 24 h treatment with high glucose. The expression of EGFP gene was （50±10）%. The expression of NG2 in RMC was significantly inhibited by Pgenesil- siNG21 and Pgenesil-siNG22. Compared with the control group, cell proliferation levels were markedly decreased in siNG2 following high glucose stimulation （P〈0.01）. Conclusion shRNA targeting NG2 gene can inhibit the cell proliferation of RMC induced by high glucose stimulation.