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

• 作 者: 隗玥 林晨 张雪艳 程金科 邢嵘 吴晣
• 机 构: 中国医学科学院中国协和医科大学肿瘤研究所分子肿瘤学国家重点实验室,中国医学科学院中国协和医科大学肿瘤研究所分子肿瘤学国家重点实验室,中国医学科学院中国协和医科大学肿瘤研究所分子肿瘤学国家重点实验室,中
• 出 处: 中华医学杂志
• 发表日期: 1999年08期
• 关键词: 细胞生长 G1期阻滞 c-myc 反义RNA 脱噬作用 细胞周期 腺病毒.人 人肺腺癌细胞系 腺病毒介导 基因表达

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?

Objective To construct recombinant adenoviruses that mediate antisense cmyc and study its effects on cell cycle, proliferation and apoptosis of human lung adenocarcinoma cells. Methods The cmyc gene was cloned into the shuttle plasmid vector p Ad C M V . The 293 cells were co-transfected with p J M17 and the recombinant adenovirus encoding antisense cmyc was generated by homologous recombination. In vitro observation and detection of recombinant adenovirus-infected human lung adenocarcinoma cell lines G L C-82 and S P C A A 1 and human embryo lung diploid cells 2 B S, cell cycle changes, apoptosis and Changes in the expression of related genes; In vivo observations on tumor cell tumorigenicity and growth of subcutaneous xenografts in nude mice. Results The recombinant adenovirus AdA Smyc coding antisense cmyc was constructed, amplified and purified. The titer reached 33×1010 plaque forming units (pfu)/ml. After tumor cells were infected with Ad-A-Smyc for 3 to 5 days, reverse transcription polymerase chain reaction (RT-RPR) and Western Blot analysis showed that the expression of c-myc gene was down-regulated. At the same time, there was obvious G1 arrest and apoptosis. In G1 phase, the expression of cyclin D1 was down-regulated and the expression of apoptosis-related genes bcl2 and bax were down-regulated and up-regulated, respectively. No changes were observed in human embryonic lung diploid cells infected with AdA Smyc. In vitro colony formation of tumor cells and growth of subcutaneous xenografts in nude mice were inhibited by AdA Smyc. Conclusion Antisense?