AFP增强型四元复合体介导N-ras反义RNA抑制HepG2.2.15细胞成瘤性的研究

Suppression of tumorigenicity of HepG2.2.15 cells by N-ras antisense RNA transfected with AFP-enhancing four-element complex

目的 观察AFP增强型四元复合体介导的N ras反义RNA转移系统对HBV转基因肝癌细胞系HepG2 .2 .15致瘤性的体内外抑制作用。方法 构建含有N ras反义RNA的AFP增强型四元复合体 ,体外瞬时转染HBV转基因HepG2 .2 .15细胞 ,流式细胞术检测转染前后N ras蛋白表达水平、细胞凋亡率及细胞周期的变化 ,同时建立稳定表达N ras反义RNA的肝癌细胞系HepG2 .2 .15 /as ras,绘制转染前后生长曲线。体内抑瘤试验分别以HepG2 .2 .15或HepG2 .2 .15 /as ras细胞制备荷瘤裸鼠模型 ,比较二者成瘤率。HepG2 .2 .15成瘤组局部瘤内注射N ras反义RNA四元复合体 ,研究其对肿瘤的抑制作用。结果 AFP增强型四元复合体介导N ras反义RNA体外瞬时转染HepG2 .2 .15细胞可显著降低胞内N ras蛋白的表达水平 (P <0 .0 5 ) ,使细胞生长停滞于G0 /G1期 ,且可诱导细胞凋亡。体内抑瘤实验显示 ,HepG2 .2 .15 /as ras细胞注射组成瘤率 ( 4 0 % )显著低于HepG2 .2 .15细胞注射组( 10 0 % )。瘤内注射N ras反义RNA四元复合体可使肿瘤体积明显缩小。结论 AFP增强型四元复合体介导的N ras反义RNA转移系统可降低HBV转基因肝癌细胞HepG2 .2 .15N ras蛋白的表达水平 ,逆转其恶性行为 。

Objective To observe the inhibitory effects of N-ras antisense RNA delivered by AFP-enhancing four-element complex on tumorigenicity of HBV-transgenic hepatocellular carcinoma cell line HepG 2.2.15 both in vitro and in vivo . Methods AFP-enhancing four-element complex,containing N-ras antisense RNA, was constructed and transfected into HepG2.2.15 cells. Seventy-two hours later, cells were collected and assayed for expression of N-ras protein, cell cycle distribution and apoptosis rate by flow cytometry. In addition, HepG 2.2.15 /as-ras,stably expressing N-ras antisense RNA, was established. The growth curve of two cell lines was delineated. BALB/c nude mice were implanted with HepG2.2.15 or HepG2.2.15/as-ras cells. The tumor-forming rate for two groups were calculated. BALB/c nude mice bearing with HepG2.2.15 cells was injected with AFP-enhancing four-element complex including N-ras antisense RNA and the volumes of tumor were measured. Results After transfection by N-ras antisense RNA four-element complex, the expression of N-ras protein of HepG2.2.15 cells was greatly decreased; cell cycle was trapped at G 0/G 1 phase; apoptosis rate was enhanced. Compared with HepG2.2.15 cells, the tumor-forming rate of HepG2.2.15/as-ras cells was lower. When HepG 2.2.15 cells were implanted into nude mice, injection of N-ras antisense RNA four-element complex could significantly decrease the volume of tumor tissues. Conclusion AFP-enhancing four-element complex including N-ras antisense RNA inhibited the expression of N-ras protein and effectively impaired the tumorigenicity of HepG2.2.15 cells both in vitro and in vivo .