腺病毒介导的反义c-myc选择性诱导肿瘤细胞G1期阻滞和凋亡的研究

目的 构建介导反义ｃ－ｍｙｃ的重组腺病毒，研究其对人肺腺癌细胞的细胞周期，增殖和凋亡的影响。方法 将ｃ－ｍｙｃ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ｃ－ｍｙｃ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；体内观察对肿瘤

Adenovirus transferred antisense c myc selectively induces tumor cell cycle arrest and apoptosis

OBJECTIVES: To construct the recombinant adenovirus encoding antisense c-myc fragment and to investigate its effect on tumor cell proliferation and apoptosis. METHODS: The shuttle plasmid encoding antisense c-myc was constructed by cloning c-myc cDNA fragment in the reverse direction into the pAdCMV. Then the plasmid pJM17 and the shuttle plasmid were co-transferred into 293 cells with liposome for homologous recombination to acquire recombinant adenovirus. The cell cycle, apoptosis, and the expression of related genes of the in vitro transferred human adenocarcinoma cell lines GLC-82 and SPC-A-1, as well as the human embryonic diploid lung cell line 2BS were studied. The effects on colony formation and treating effect on transplanted tumor in nude mice were also studied. RESULTS: The recombinant adenovirus encoding antisense c-myc fragment was obtained with the titer of 3.3 x 10(10) pfu/ml. RT-PCR and Western blot showed that the expression of c-myc was reduced 3-5 days after infection. Simultaneously, obvious G1 arrest and apoptosis as well as the alteration of cyclin D1, bcl-2 and bax gene expression were observed in the infected tumor cells. The infected 2BS cells showed no such changes. The colony formation ability in vitro and the growth of the hetero-transplanted tumor in nude mice were also reduced. CONCLUSIONS: The reduced expression of c-myc gene could induce human adenocarcinoma cells tumor-cell-specific G1 arrest and apoptosis, while it has no effect on normal cell.