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目的:构建介导反义cyclin D1的重组腺病毒,研究其对人肺腺癌细胞内的细胞周期、增殖和凋亡的影响.方法:将cy-clin D1基因反向克隆于穿梭质粒载体pAdCMV中,通过脂质体与pJM17共转染293细胞,经同源重组产生编码反义cyclin D1的重组腺病毒.体外观察和检测重组腺病毒感染人肺腺癌细胞系GLC-82和SPC-A-1以及人胚肺二倍体细胞2BS后,细胞周期的改变、凋亡的发生和相关基因表达的变化.结果:构建并扩增、纯化得到编码反义cyclin D1的重组腺病毒Ad-AS cyclin D1,滴度达1×10~(10)pfu/ml.肿瘤细胞感染Ad-AS cyclin D1后,RT-PCR分析显示cyclin D1基因表达下调;同时出现了明显的G1期阻滞和凋亡,凋亡相关基因bcl-2和bax基因的表达分别出现下调和上调;人胚肺二倍体细胞感染Ad-ASmyc后无上述变化.肿瘤细胞体外克隆形成能力Ad-AS cyclin D1抑制.结论:反义cyclin D1的重组腺病毒感染肿瘤细胞可使cyclin D1基因表达下调,继而诱导肿瘤细胞G1期阻滞和凋亡,使肿瘤细胞致瘤能力和肿瘤生长被抑制,但不影响正常细胞.
OBJECTIVE: To construct a recombinant adenovirus that mediates antisense cyclin D1 and investigate its effect on cell cycle, proliferation, and apoptosis in human lung adenocarcinoma cells. Methods: Reverse cloning of cy-clin D1 gene into shuttle plasmid vector pAdCMV In vitro, 293 cells were cotransfected with liposome and pJM17, and the recombinant adenovirus encoding antisense cyclin D1 was generated by homologous recombination. In vitro observation and detection of recombinant adenovirus infected human lung adenocarcinoma cell line GLC-82 and SPC-A -1 and changes in cell cycle, apoptosis, and expression of related genes after human embryonic lung diploid cell 2BS. Results: Construction and amplification, purification of recombinant adenovirus encoding antisense cyclin D1 Ad-AS Cyclin D1 with a titer of 1×10 10 pfu/ml. After tumor cells were infected with Ad-AS cyclin D1, RT-PCR analysis showed down-regulation of cyclin D1 gene expression; obvious G1 arrest and apoptosis were also observed. The expression of apoptosis-related genes bcl-2 and bax were down-regulated and up-regulated, respectively; the above changes were not observed in human embryonic lung diploid cells infected with Ad-ASmyc. The in vitro colony-forming ability of tumor cells was inhibited by Ad-AS cyclin D1. Antisense cyclin D1 recombinant adenovirus infects tumor cells to make cyclin D1 gene Up to down, in turn induce arrest and apoptosis of tumor cells in G1 phase, tumorigenicity of tumor cells and tumor growth is inhibited without affecting normal cells.