反义c-myc基因转染抑制人晶状体上皮细胞增殖的研究

目的探讨腺病毒介导的反义cmyc基因(AdASmyc)转染对人晶状体上皮细胞(HLEC)系B3(HLEB3)增殖、凋亡及细胞周期的影响。方法将AdASmyc转染HLEB3,观察细胞形态变化;采用生长曲线法、噻唑蓝比色法分析细胞增殖改变;使用流式细胞仪测定细胞凋亡变化、分析细胞周期改变(DNA倍体法);采用逆转录聚合酶链反应(RTPCR)法、Western免疫印迹法检测细胞cmycmRNA及其蛋白产物表达的变化。结果AdASmyc成功转染后,HLEB3逐渐变圆、脱壁。转染后24~96h细胞的增殖受到抑制,与相应对照组比较,差异均有统计学意义(P<001);凋亡细胞比例(转染48h为1833%,转染96h为2693%)明显升高,与相应对照组(48h为319%,96h为175%)比较,差异均有统计学意义(P<001);细胞出现G1期阻滞现象,表现为G1期细胞比例转染48h为(6050±759)%,转染96h为(8190±860)%]增加和S期细胞比例转染48h为(2840±338)%,转染96h为(1675±897)%]下降,与相应对照组比较,差异均有统计学意义(P<001);RTPCR法和Western免疫印迹法检测发现cmycmRNA及其蛋白产物水平特异性下调。结论AdASmyc可成功转染HLEB3,并特异性抑制cmyc基因的表达,诱发细胞G1期阻滞,有效抑制细胞增殖,诱导细胞凋亡。

Inhibition of proliferation of human lens epithelial cell line HLE-B3 by adenovirus-mediated transfer of antisense c-myc gene

OBJECTIVE: To investigate the effects of adenovirus-mediated transfer of antisense c-myc gene on proliferation and survival of human lens epithelial cell. METHODS: Immortalized human lens epithelial cell line (HLE-B3) was infected by replication-deficient adenovirus of antisense c-myc gene (Ad-AS-myc). The expression of c-myc mRNA and protein was detected by RT-PCR and Western blotting respectively. Cell proliferation was analyzed by cell growth curve and MTT colorimetric assay, cell death and cell cycle of HLE-B3 were examined by flow cytometry. RESULTS: More than 80% of HLE-B3 cells were infected successfully after 48 hours incubation with Ad-AS-myc adenovirus. The expression of c-myc mRNA and protein was decreased. Ad-AS-myc suppressed the growth rate, enhanced the cell death (18.33% after 48 hours and 26.93% after 96 hours transduction). The proportions of G1 phase cells were increased to (60.50 +/- 7.59)% after 48 hours and (81.90 +/- 8.60)% after 96 hours transduction, while the proportions of S phase cells were decreased to (28.40 +/- 3.38)% after 48 hours and (16.75 +/- 8.97)% after 96 hours infection. CONCLUSIONS: Adenovirus-mediated antisense c-myc may inhibit proliferation and induce apoptosis of HLE-B3.