Caspase-3在Apoptin基因诱导Hela细胞凋亡中的作用  

Role of Caspase-3 in Apoptin-induced apoptosis of Hela cells

作　　者：曹婧[1] 赵洪礼[2] 黄敏[1] 戈全治[2] 宁安红[1] 

机构地区：[1]大连医科大学微生物学教研室,大连116027 [2]沈阳军区联勤部疾病预防控制中心分子生物学实验室,沈阳110034

出　　处：《中国肿瘤生物治疗杂志》2007年第4期333-337,共5页Chinese Journal of Cancer Biotherapy

基　　金：辽宁省自然科学基金资助课题(20042046)~~

摘　　要：目的:研究Caspase-3在Apoptin gene诱导Hela细胞凋亡中的作用。方法:用含有Apoptin基因的真核表达载体pcDNAA3瞬间转染体外培养的Hela细胞;Hela细胞瞬间转染后0、24、48、72和96h,分为正常的Hela细胞对照组,pcD-NA3.1质粒转染对照组和实验组。分别采用RT-PCR、DNA凝胶电泳、流式细胞术检测Hela细胞的凋亡;以比色法检测Caspase-3的相对活性。结果:以转染后24、48和72hHela细胞的总RNA为模板,经RT-PCR均可扩增出Apoptin cDNA,正常Hela细胞和空载体转染细胞均为阴性表明Apoptin已在转染细胞中表达;Apoptin基因瞬间转染的Hela细胞可出现典型的细胞凋亡所具有的DNA梯状带,表明有凋亡发生;FCM检测发现,以Apoptin基因转染后48h,在DNA直方上的G1峰前,即出现1个明显的亚2倍体峰(亚G1峰,即凋亡峰),并随着转染时间延长而增强。细胞凋亡率与对照组相比较差异显著(P<0.05)。Hela细胞经pcDNAA3质粒转染后24h实验组Caspase-3的活性开始升高,72h达高峰,明显高于正常细胞对照组和pcDNA3.1对照组(P<0.01)。结论:Apoptin基因可通过激活Caspase-3诱导Hela细胞凋亡。Objective： To explore the role of Caspase-3 in Apoptin gene-induced apoptosis of Hela cells. Methods： Hela cells were transiently transfeeted with recombinant pcDNAA3 plasmid containing Apoptin gene. This study was divided into normal control group, pcDNA3.1 transfected group, and experimental group. The apoptoses of Hela cells were measured by DNA agarose eleetrophoresis, RT-PCR and flow eytometry. The relative activity of Caspase-3 was determined by eolorimetrie assay instantly at 24, 48, 72, and 96 h after transfeetion. Results ：The eDNA of Apoptin gene was detected by RT-PCR in the total RNA of the Hela cells transfeeted with peDNAA3 at 24, 48 and 72 h after transfeetion, but not in that of normal Hela cells and peDNA3. 1 transfeeted Hela cells. The DNA ladder, indicator of apoptosis cells, was found in Hela cells transfected with pcDNAA3 at 24, 48 and 72 h after transfection. FCM analysis found that the sub-G1 peak （ apoptosis peak） in the graph of Hale cells transfected with peDNAA3 at 48 h. These results showed that Apoptin gene indueed apoptosis in Hela eells in vitro ; the apoptosis rate was significantly higher than that of control group as time went by. Caspase-3 activity began to rise at 24 h and reached the peak at 72 h in Hela cells after peDNAA3 transfeetion, but not in normal Hela cells and peDNA3.1 transfeeted Hela cells. Conclusion： Apoptin gene can induce apoptosis of Hela cells v/a activation of Caspase-3.

关 键 词：肿瘤特异性凋亡基因 凋亡 CASPASE-3 基因治疗 

分 类 号：R459.9[医药卫生—治疗学]