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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 investigate the effect of survivin antisense oligonucleotide (survivin-ASODN) on the apoptosis of bladder cancer cell line BIU87 induced by chemotherapeutic drug docetaxel (Taxotere). Methods The constructed survivin-ASODN eukaryotic expression vector pcDNA3-SVVas was transfected into bladder cancer cell line BIU87 by lipofectamine 2000. The positive clones transfected with the recombinant plasmid were screened by reverse transcriptase-polymerase chain reaction (RT- PCR was used to detect the expression of survivin mRNA. The effect of survivin-tasdin combined with taxotere on the survival of BIU87 cells was observed by trypan blue exclusion staining. The cell counts and MTT assay The sensitivity of cells to taxotere was analyzed. The DNA fragmentation of apoptotic cells was analyzed by agarose gel electrophoresis. The changes of nuclei of apoptotic cells were detected by nuclear staining. The apoptosis rate was detected by flow cytometry. Results BIU87-SVVas cells transfected survivin-ASODN eukaryotic expression vector pcDNA3-SVVas expression of survivin mRNA levels decreased, cell proliferation was significantly inhibited, and transfected with pcDNA3 empty vector BIU87-neo cells, untransfected vector BIU87 cells (All P <0.05). DNA ladder was observed in BIU87-SWas cells by agarose gel electrophoresis, but not in other control groups. Compared with BIU87-neo and BIU87 cells The cell nuclei of BIU87-SVVas cells were densely stained. The apoptosis rate of BIU87-SWas cells treated with Taxotere increased significantly. Conclusions Survivin-ASODN can promote the apoptosis of BIU87 cells induced by Taxotex and increase its sensitivity to taxotere, which lays an experimental foundation for the biological treatment of bladder cancer.