衔接蛋白p66Shc在高糖诱导的肾小管上皮细胞线粒体活性氧簇产生中的作用  

Effect of Adaptor Protein p66Shc on Mitochondrial ROS Overproduction Induced by High Glucose in Human Renal Proximal Epithelial Cells

作　　者：聂静[1] 孙林[1] 刘伏友[1] 刘映红[1] 孙岩[1] 邹莎琳[1] 

机构地区：[1]中南大学湘雅二医院肾内科,长沙410011

出　　处：《中国中西医结合肾病杂志》2009年第9期757-760,I0001,共5页Chinese Journal of Integrated Traditional and Western Nephrology

基　　金：湖南省自然科学基金资助项目(No.09JJ6031)

摘　　要：目的:观察高糖环境下人近端肾小管上皮细胞HK-2中p66Shc的表达和磷酸化,以及p66Shc瞬时高表达对高糖诱导的HK-2线粒体活性氧簇产生的影响,为探讨糖尿病肾病线粒体ROS产生的机制提供新的思路。方法:常规培养正常人近端肾小管上皮细胞(HK-2),将30mmol/LD-葡萄糖在不同时间点作用于体外培养的HK-2细胞,采用Real-time-PCR检测p66ShcmRNA的表达变化,Westem blot检测p66Shc蛋白、磷酸化p66Shc-Ser36蛋白的表达;进一步将pcDNA3.1hisp66Shc质粒转染HK-2细胞,采用激光共聚焦显微镜观察p66Shc高表达对高糖诱导的线粒体活性氧簇产生的影响。结果:30mmol/LD-葡萄糖能呈时间依赖性上调p66Shc mRNA和蛋白表达水平,同时增强p66Shc第36位丝氨酸的磷酸化;在30mmol/LD-葡萄糖作用下,转染p66Shc的HK-2线粒体ROS水平明显高于未转染组及空载体转染组(P<0.05)。结论:p66Shc参与了高糖诱导的线粒体活性氧簇的产生,可能在糖尿病肾病早期肾小管氧化损伤过程中发挥重要作用。Objective： To examine the expression of p66Shc and the phosphorylation of p66shc - Ser36 in the treatment with high glucose in HK - 2, and to investigate the effect of p66Shc over expression on mitochondrial ROS production induced by high glucose. Methods：HK- 2 cells were treated with D- glucose （30 mmol/L） in different time points. The expression of p66Shc mRNA was examined by Real time PCR. Expressions of p66Shc and phospho- Ser36 protein were detected by Western blot. The plamid pcDNA 3.1 hisp66She was transfected into HK - 2 before exposed to high glucose, and changes of mitochondrial ROS was detected with confocal microscope. Results： Stimulation of HK - 2 with 30 mM D - glucose resulted in a time dependent increase in the expression and phosphorylation of p66Shc. The level of mitochondrial ROS induced by high glucose showed an obvious differences between the p66Shc ＋ 30 mmol/L group and 30 mmol/L group or pcDNA3.1 ＋ 30 mmol/L group （P 〈 0.05）. Conelusion：p66Shc may be involved in the pathogenesis of mitochondrial ROS overproduction induced by high glucose and play a key role in tubular oxidative injury during early stage of diabetic nephropathy.

关 键 词：葡萄糖 近端肾小管上皮细胞 活性氧簇 

分 类 号：R587.2[医药卫生—内分泌]