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

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，Western 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p66ShcmRNA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。

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

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 hisp66Shc was transfected into...