Gli2 对高糖条件下肾小管上皮细胞凋亡及ROS 水平影响

目的:探讨胶质瘤相关癌基因2(Gli2)对高糖条件下的肾小管上皮细胞凋亡及活性氧(ROS)水平影响。方 法:用肾小管上皮细胞NRK鄄52E 为研究对象,分别转染Gli2 过表达载体和对照载体,同时用不转染的细胞作为对照,RT鄄PCR 和Western blot 检测Gli2 水平,用高糖和低糖细胞培养液培养不转染的对照细胞,同时以高糖培养液培养转染Gli2 过表达载 体的细胞,流式细胞术检测细胞凋亡,二氯二氢荧光素二乙酸酯(DCFH-DA)探针法检测ROS 含量,黄嘌呤氧化酶法检测超氧 化物歧化酶(SOD)含量,Western blot 检测Bcl-2 相关X 蛋白(Bax)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、Ptch 相关跨膜蛋白Smoothened(Smo)水平。结果:NRK-52E 细胞转染Gli2 过表达载体后的Gli2 mRNA 和蛋白水 平均明显高于对照细胞(P<0.01),而转染对照载体后的NRK鄄52E 细胞中Gli2 mRNA 和蛋白水平与对照细胞相比没有差异 (P>0.05)。高糖培养后的不做转染的NRK鄄52E 细胞凋亡率升高,ROS 含量升高,SOD 含量下降,细胞中Bax、Cleaved Caspase- 3 水平升高,Smo 水平下降,与对照细胞相比差异具有统计学意义(P<0.01)。而过表达Gli2 后的NRK-52E 细胞经高糖培养 后,细胞凋亡率下降,ROS 含量降低,SOD 含量升高,细胞中Bax、Cleaved Caspase-3 水平下降,Smo 水平升高,与单纯高糖培养 的NRK-52E 细胞相比,差异具有统计学意义(P<0.01)。结论:高糖诱导肾小管上皮细胞凋亡和氧化损伤,Gli2 可以降低高糖 诱导的肾小管上皮细胞凋亡,减轻氧化损伤。

Effect of Gli2 on apoptosis and ROS level in renal tubular epithelial cells under#br# high glucose

Objective:To investigate the effect of Gli2 on apoptosis and ROS level in renal tubular epithelial cells under high glucose.Methods:The renal tubular epithelial cell NRK-52E was used as the object of study,Gli2 overexpression vector and control vector were transfected,the non transfected cells were also used as controls,RT-PCR and Western blot detected Gli2 levels,culture of non transfected cells by high glucose and low glucose cell culture medium,cells transfected with Gli2 overexpression vector were cultured in high glucose medium,apoptosis was detected by flow cytometry,the ROS content was detected by DCFH-DA probe method, the content of SOD was detected by xanthine oxidase method;Bax,Cleaved Caspase-3 and Smo levels were detected by Western blot.Results:The Gli2 mRNA and protein levels of NRK-52E cells transfected with Gli2 overexpression vector were significantly higher than that of control cells(P<0.01),the Gli2 mRNA and protein levels in the NRK-52E cells transfected with the control vector were not different from those of the control cells(P>0.05).The apoptosis rate of NRK-52E cells without transfection was elevated after high glucose culture,elevated ROS content,SOD content decreased,the levels of Bax,Cleaved Caspase-3 were elevated in the cells,Smo levels declined,compared with the control cells,the difference was statistically significant(P<0.01).After overexpression of Gli2 over- expression vector,NRK-52E cells were cultured with high glucose,the rate of apoptosis decreased,ROS content decreased,elevated SOD content,the levels of Bax,Cleaved Caspase-3 decreased,elevated levels of Smo,compared with NRK-52E cells cultured with simple high glucose,the difference was statistically significant(P<0.01).Conclusion:Apoptosis and oxidative damage in renal tubular epithelial cells induced by high glucose,Gli2 can decrease the apoptosis of renal tubular epithelial cells induced by high glucose,mitigate oxidative damage.