Clusterin抑制人肾近曲小管上皮HK-2细胞的凋亡

Clusterin是一种硫酸糖蛋白.最近研究发现，clusterin具有抗凋亡作用，同时对肾细胞具有保护作用，但抗凋亡的具体机制仍不清楚.为研究clusterin及其不同功能区域在人肾近曲小管上皮HK-2细胞中的抗凋亡作用，构建了含有全长及缺失前导序列的clusterin重组质粒（分别命名为pIRES2-EGFP/cluac和pIRES2-EGFP/clubc）.将重组质粒转染人肾近曲小管上皮HK-2细胞后，检测转染细胞中clusterin的表达及其抗Na₂SeO₃（10μmol/L）诱导的凋亡作用.Western印迹显示，转染pIRES2-EGFP/cluac的HK-2细胞培养上清及细胞裂解液中均可检测到clusterin蛋白表达，但转染pIRES2-EGFP/clubc的HK-2细胞仅在裂解液中检测到clusterin，在培养上清液中未检测到该蛋白表达.流式细胞术检验显示，HK-2 /clubc细胞实验组出现明显凋亡峰，而 HK-2 /cluac细胞组则未见凋亡；两组的凋亡百分率之间也存在显著性差异（P<0.05）.以Cy3标记的Annexin V染色后于荧光显微镜下观察细胞凋亡情况与FCM检测结果基本一致.上述结果证明，clusterin有明显的抑制人肾近曲小管上皮HK-2细胞凋亡的作用；clusterin前导序列是其发挥抗凋亡作用的必需功能区域，提示clusterin抗凋亡作用是通过细胞外途径产生的.

Clusterin Prevents Apoptosis in Human Proximal Tubular Cells HK-2

Clusterin is a sulfated glycoprotein and prevents apoptosis in kidneycells. To evaluate the effect of clusterin containing different domains , the mechanism of preventing apoptosis was studied when the different domains of clusterin were over-expressed in human proximal tubular cells (PTC) HK-2. The plasmid pIRES2-EGFP was used to generate the clusterin expression constructs with full length or without the leader sequence (designated as pIRES2-EGFP/cluac, pIRES2E-GFP/clubc, respectively). Western blotting was employed to compare clusterin protein expression levels in the lysate and supernatant of HK-2 cells which were transfected by clusterin. When HK-2 cells were induced to produce by Na₂SeO₃, the clusterin’s protective role was examined by flow cytometry (FCM) and fluorescence microscope in HK-2 cells. Clusterin expression was detected in the lysate andsupernatant of pIRES2-EGFP/cluac transfected HK-2 cells; while clusterin was only found in lysate of pIRES2-EGFP/clubc transfected HK-2 cells, whereas it was not found in supernatant. Its anti-apoptotic property in HK-2 cells was confirmed by FCM and fluorescence microscope. It was apparent that clusterin played an important role in preventing apoptosis in human proximal tubular cells, and the presence of the leader sequence was necessary for clusterin’s anti-apoptotic function.