Xin Dian-qi, Zhu Xu-hui, Ai Jun-kui, Na Yan-qun, Guo Ying-lu
Department of Urology, Peking University First Hospital, Beijing 100034, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2004 Apr;36(2):154-8.
To evaluate the effect of clusterin with and without leader sequence on overexpression preventing apoptosis in human prostate LNCaP cells.
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, pIRES2-EGFP/clubc, respectively). Western blot analysis was employed to compare clusterin expression levels in the lysis and supernatant fluid of clusterin transfected LNCaP cells in vitro. The distribution of different functional domains of clusterin in cells was detected with Immunocytochemical staining. The clusterin's protective role of Na2SeO3-induced apoptosis in LNCaP cells was examined by flow cytometry (FCM) and fluorescence microscope.
Clusterin expression was detected in the lysis and supernatant fluid of pIRES2-EGFP/cluac transfected LNCaP cells, while clusterin was found only in lysis liquid of pIRES2-EGFP/clubc transfected LNCaP cells, but not found in their supernatant fluid. The distribution of cluserin in the plasm of pIRES2-EGFP/cluac transfected cells was aggregative, and on the other hand, clusterin distributed dispersedly in pIRES2-EGFP/clubc transfected cells. Its anti-apoptotic property in LNCaP cells was proved by FCM and fluorescence microscope.
It is apparent that clusterin plays an important role in preventing apoptosis in prostate cancer, and the presence of the leader sequence is necessary for clusterin's anti-apoptotic function.
评估有无前导序列的簇集蛋白过表达对人前列腺LNCaP细胞凋亡的抑制作用。
利用质粒pIRES2-EGFP构建全长或无前导序列的簇集蛋白表达载体（分别命名为pIRES2-EGFP/cluac、pIRES2-EGFP/clubc）。采用蛋白质免疫印迹分析比较体外转染簇集蛋白的LNCaP细胞裂解液和上清液中簇集蛋白的表达水平。通过免疫细胞化学染色检测簇集蛋白不同功能域在细胞内的分布。运用流式细胞术（FCM）和荧光显微镜检测簇集蛋白对亚硒酸钠诱导的LNCaP细胞凋亡的保护作用。
在转染pIRES2-EGFP/cluac的LNCaP细胞的裂解液和上清液中均检测到簇集蛋白表达，而在转染pIRES2-EGFP/clubc的LNCaP细胞中，仅在裂解液中发现簇集蛋白，在上清液中未检测到。在转染pIRES2-EGFP/cluac的细胞胞质中，簇集蛋白呈聚集分布，而在转染pIRES2-EGFP/clubc的细胞中，簇集蛋白呈分散分布。流式细胞术和荧光显微镜证实了簇集蛋白在LNCaP细胞中的抗凋亡特性。
显然，簇集蛋白在前列腺癌细胞凋亡抑制中发挥重要作用，且前导序列的存在对于簇集蛋白的抗凋亡功能是必需的。
