摘要
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 the recombinant adenovirus encoding antisense cyclin Dl fragment, and to investigate its effect on tumor cell proliferation and apoptosis. Methods: The shutde plasmid encoding antisense cyclin Dl was constructed by cloning cyclin Dl cDNA fragment in the reverse direction into .the pAdCMV. Then the plasmid pJM17 and the shutde plasmid were co-transferred into 293 cells with liposome for homologous recombination to acquire recombinant adenovirus. The cell cycle, apoptosis, and the expression of the related genes of the in vitro transferred human adenocar-cinoma cell lines GLC-82 and SPC-A-1, as well as the human embryonic diploid lung cell line 2BS were studied. The effect on colony formation was also studied. Results: The recombinant adenovirus encoding antisense cyclin Dl fragment Ad-AS cyclin Dl was obtained with the liter of 1 x 10^(10) pfu/ml. RT-PCR assay showed that the expression of cyclin Dl of GLC-82 and SPC-A-1 was reduced after Ad-AS cyclin Dl infection. Simultaneously, obvious Gl arrest and apoptosis, bcl-2 and bax gene expression changes were observed in the infected tumor cells. The infected 2BS cells showed no such changes. The colony formation ability in vitro was also reduced. Conclusion; The reduced expression of cyclin Dl gene could induce human lung adenocarcinoma cells specific Gl arrest and apoptosis, while has no effect on normal cell.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
1999年第2期91-96,共6页
Chinese Journal of Cancer Biotherapy
基金
"863"计划项目Z20-01-02资助
关键词
腺病毒
反义cyclinD1
腺癌
细胞周期
肺肿瘤
adenovirus
antisense cyclin Dl
lung adenocarcinoma
cell cycle
cell growth
apoptosis
