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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 is a sulfuric acid glycoprotein.Recent studies have found that clusterin has anti-apoptotic effects, while having a protective effect on renal cells, but the specific mechanism of anti-apoptotic remains unclear.In order to study clusterin and its different functional areas in human kidney near The anti-apoptotic effect of clusterin on HK-2 cells was detected by PCR and the clusterin recombinant plasmids containing pIRES2-EGFP / cluac and pIRES2-EGFP / clubc were constructed.The recombinant plasmids were transfected into human The expression of clusterin in transfected cells and the apoptosis induced by anti-Na2SeO3 (10μmol / L) were detected by Western blotting.The HK-2 cells transfected with pIRES2-EGFP / cluac Clusterin protein expression was detected in both culture supernatants and cell lysates, but clusterin was detected only in lysates transfected with pIRES2-EGFP / clubc-HK-2 cells, and no detectable protein expression in culture supernatants Flow cytometry showed that there was a significant apoptosis peak in HK-2 / clubc cells in experimental group, but no apoptosis in HK-2 / cluac cells group; there was also a significant difference between the two groups P <0.05). Cy3-labeled Annexin V stained fluorescence The results of microdialysis showed that the cell apoptosis was basically the same as that of FCM, which indicated that clusterin could obviously inhibit the apoptosis of HK-2 cells in human renal proximal tubule epithelial cells. Clusterin leader was the anti-apoptotic Necessary functional areas suggest that anti-apoptotic effects of clusterin are produced by extracellular pathways.