生存素反义寡核苷酸诱导膀胱癌细胞凋亡及增强其化疗敏感性  

Antisense RNA of survivin gene enhances the taxol-induced apoptosis and sensitivity to chemotherapy drugs in bladder cancer cells:an in vitro experiment

作　　者：王志华[1] 叶庆[2] 黄德樱[3] 胡志全[1] 叶章群[1] 阳诺[1] 刘昊[1] 庄乾元[1] 杨为民[1] 刘继红[1] 张旭[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院泌尿外科,武汉430030 [2]华中科技大学同济医学院免疫学系 [3]华中科技大学同济医学院附属武汉市妇女儿童医疗保健中心

出　　处：《中华医学杂志》2007年第6期419-422,共4页National Medical Journal of China

基　　金：国家自然科学基金(30070325)

摘　　要：目的探讨生存素反义寡核苷酸(生存素-ASODN)对化疗药多西紫杉醇(泰索帝)诱导膀胱癌细胞系 BIU87细胞凋亡的影响。方法将已构建成功的生存素-ASODN 真核表达载体pcDNA3-SVVas 通过脂质体介导转染膀胱癌细胞系 BIU87,并筛选转染成功的阳性克隆;采用逆转录聚合酶链反应(RT-PCR)法检测其生存素 mRNA 表达;锥虫蓝拒染法观察生存素-ASODN 与泰索帝联合应用对 BIU87细胞生存的影响;细胞计数和四甲基偶氮唑盐(MTT)试验测定转染细胞对泰索帝敏感性;琼脂糖凝胶电泳分析细胞凋亡 DNA 断裂情况;核染色检测凋亡细胞的细胞核的变化;流式细胞术检测细胞凋亡率。结果转染生存素-ASODN 真核表达载体 pcDNA3-SVVas 的 BIU87-SVVas 细胞生存素 mRNA 表达水平下降、细胞增殖明显受抑,与转染 pcDNA3空载体 BIU87-neo 细胞、未转染载体的 BIU87细胞进行比较,差异均有统计学意义(均 P<0.05);经琼脂糖凝胶电泳,BIU87-SWas 细胞可见到 DNA 梯形条带,而其他对照组未见到;与 BIU87-neo、BIU87细胞相比,BIU87-SVVas 细胞的细胞核呈致密浓染;加入泰索帝的 BIU87-SWas 细胞组的凋亡率大幅度增加。结论生存素-ASODN 可促进泰索帝诱导 BIU87细胞凋亡及增加其对泰索帝的敏感性,为膀胱癌的生物学治疗研究奠定了实验基础。Objective To evaluate the effects of antisense oligedeexynucleotide （ ASODN ） of survivin gene on taxol-inducod apoptosis in bladder cancer cells. Methods A survivin ASODN eukaryotic vector pcDNA3-SVVas was transfected into human bladder cancer cells of the line BIU87 mediated by liposomal reagent （ BIU87 SVVas cells）. The mRNA expression of survivin was measured by RT-PCR. Blank plasmid pcDNA3 was transfected as control （BIU87 neo cells）. The cell growth curve was drawn using trypan blue dye exclusion assay. MTT assay was used to investigate the sensibility of transfected cells to taxol. The BIU87 SVVas cells, BIU87 neo cells, and BIU87 cells were cultured in the culture fluid with taxol and then DNA gel electrophoresis, Hoechst nuclear staining and fluorescent microscopy, and annexin- ropidium iodide （PI） staining were used to examine the cell apoptosis. Results Compared to BIU87 and BIU87 neo cells, the mRNA expression of survivin in the BIU87-SVVas cells was obviously reduced. The growth of the BIU87-SVVas cells was significantly reduced in comparison with the BIU87 （ P 〈 0.05 ）. The sensibility of BIU87 SVVas cells to taxol increased significantly shown by cell proliferation and MTT assays. Agarose gel electrophoresis of genomic DNA showed typical DNA ladder only in the BIU87 SVVas cells, but not in other cells. Hoechst nuclear staining and fluorescent microscopy showed that the nuclei of the BIU87 SWas cells become condense. Annexin-PI test shoed that the cell apoptosis rate of the BIU87-SVVas cells treated with taxol was significantly higher than those of the other groups. Condusion survivin antisense RNA enhances the taxol-induced apoptosis in the bladder cancer cells. This may lay an experimental foundation for further research of biotherapy in bladder cancer. [

关 键 词：膀胱肿瘤 生存素 反义核酸 泰索帝 

分 类 号：R737.14[医药卫生—肿瘤]