Survivin-2B诱导人乳腺癌细胞凋亡的初步研究

摘要

目的：探讨存活蛋白2B （ survivin-2B）在诱导肿瘤细胞凋亡中的分子机制。方法：将survivin-2B基因插入真核表达载体pCDNA3．1，得到重组载体pCDNA3．1-survivin-2B。将空载体pCDNA3．1及重组载体pCD-NA3．1-survivin-2B分别转染人乳腺癌细胞株MCF7，转染后48 h，用annexin V／7-AAD染色法分析细胞凋亡情况，利用碘化丙啶染色法分析转染对细胞周期的影响；同时提取总RNA并反转录成cDNA，进行多重聚合酶链反应。利用GeXP多基因表达分析系统检测21个与肿瘤相关基因的表达情况。结果：过表达survivin-2B基因导致MCF7细胞凋亡与细胞周期阻滞，并引起8个基因表达上调，2个基因表达下调。变化最大的为醛脱氢酶4家族成员A1（ALDH4A1），表达下降48％；变化最小的为胞质分裂调控蛋白1（PRC1），上调1．08倍。结论： Survivin-2B能够诱导细胞凋亡与细胞周期G2／M期阻滞，并引起部分肿瘤相关基因的表达变化。%AIM:To explore the role of survivin-2B in the process of tumor cell apoptosis .METHODS:The survivin-2B gene was cloned into pcDNA3.1 vector and the recombinant plasmid pcDNA3.1-survivin-2B was obtained.Hu-man breast cancer MCF7 cells were transfected with pcDNA3.1 and pcDNA3.1-survivin-2B using Lipofectamine 2000.The cell cycle was determined by propidium iodide staining , and the apoptosis was detected by annexin V/7-AAD staining 48 h after transfection.Meanwhile, tatal RNA was extrated and multiplex polymerase chain reaction based on GenomeLab GeXP Genetic Analysis System was performed to detect the expression of 21 tumor-related genes .RESULTS: Flow cytometry analysis indicated that over-expression of survivin-2B promoted the apoptosis and cell cycle arrest of MCF 7 cells.Compared with control group , totally 10 differential expressed genes were related to the over-expressed survivin-2B, among which 2 were up-regulated and 8 were down-regulated. The expression of aldehyde dehydrogenase 4 family member A1 (ALDH4A1) was 48%down-regulated, and the expression of protein regulator of cytokinesis 1 (PRC1) was 1.08 folds up-regulated.CONCLUSION:Survivin-2B induces the expression changes of some tumor-related genes, which results in the apoptosis and G 2/M arrest of MCF7 cells.