摘要
目的探讨腺病毒介导的反义c-myc基因(Ad-AS-myc)转染对人晶状体上皮细胞(HLEC)系B3(HLE-B3)增殖、凋亡及细胞周期的影响. 方法将Ad-AS-myc转染HLE-B3,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(RT-PCR)法、Western免疫印迹法检测细胞c-myc mRNA及其蛋白产物表达的变化. 结果 Ad-AS-myc成功转染后,HLE-B3逐渐变圆、脱壁.转染后24～96 h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<0.01);凋亡细胞比例(转染48 h为18.33%,转染96 h为26.93%)明显升高,与相应对照组(48 h为3.19%,96 h为1.75%)比较,差异均有统计学意义(P<0.01);细胞出现G1期阻滞现象,表现为G1期细胞比例[转染48 h为(60.50±7.59)%,转染96 h为(81.90±8.60)%]增加和S期细胞比例[转染48 h为(28.40±3.38)%,转染96 h为(16.75±8.97)%]下降,与相应对照组比较,差异均有统计学意义(P<0.01);RT-PCR法和Western免疫印迹法检测发现c-myc mRNA及其蛋白产物水平特异性下调. 结论 Ad-AS-myc可成功转染HLE-B3,并特异性抑制c-myc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.(中华眼科杂志,2005,41:161-165)
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.(Chin J Ophthalmol,2005,41:161-165)
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2005年第2期161-165,共5页
Chinese Journal of Ophthalmology
基金
山东省自然科学基金资助项目(Y2003C04)
