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

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

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ｐ Ａｄ Ｃ Ｍ Ｖ 中，通过脂质体与ｐ Ｊ Ｍ１７ 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 Ｇ Ｌ Ｃ８２ 和 Ｓ Ｐ Ｃ Ａ１ 以及人胚肺二倍体细胞２ Ｂ Ｓ后，细胞周期的改变、凋亡的发生和相关基因表达的变化；体内观察对肿瘤细胞致瘤能力和裸鼠皮下移植瘤生长的影响。结果 构建并扩增、纯化得到编码反义ｃｍｙｃ 的重组腺病毒 Ａｄ Ａ Ｓｍｙｃ，滴度达３３ ×１０１０ 噬斑形成单位（ｐｆｕ）／ｍｌ。肿瘤细胞感染 Ａｄ Ａ Ｓｍｙｃ ３ ～５ 天后，逆转录聚合酶链反应（ Ｒ Ｔ Ｐ Ｃ Ｒ） 和 Ｗｅｓｔｅｒｎ Ｂｌｏｔ 分析显示ｃｍｙｃ 基因表达下调；同时出现了明显的 Ｇ１ 期阻滞和凋亡， Ｇ１ 期正调控基因ｃｙｃｌｉｎ Ｄ１ 表达下调，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 Ａ Ｓｍｙｃ 后无上述变化。肿瘤细胞体外克隆形成能力和裸鼠皮下移植瘤生长被 Ａｄ Ａ Ｓｍｙｃ 抑制。结论 反义?

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 3×10 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