腺病毒介导凋亡素诱导人大细胞肺癌NCI-H460细胞凋亡的研究

Apoptosis-inducing effect of adenovirus-mediated Apoptin on human large cell lung cancer NCI-H460 cells

目的:构建携带VP3基因重组腺病毒载体(p Adxsi-GFP-VP3),探讨凋亡素(Apoptin)在人大细胞肺癌NCI-H460细胞中的表达及诱导凋亡效应。方法:以pc DNA3.1-VP3重组质粒为模板,经酶切将VP3基因连接至穿梭质粒,后转移至p Adxsi载体上,收获p Adxsi-GFP-VP3,经酶切和测序鉴定正确后进行病毒扩增和纯化,最后测定病毒滴度。将p Adxsi-GFP-VP3以最适感染复数转染入人大细胞肺癌NCI-H460细胞,利用RT-PCR(reverse transcription polymerase chain reaction,RT-PCR)和Western blot技术检测Apoptin在细胞中的表达情况;透射电镜(transmission electron microscopy,TEM)下观察细胞的超微结构;MTT法检测细胞的增殖活性;流式细胞仪(flow cytometry,FCM)检测转染后细胞的凋亡率及细胞周期。结果:成功构建重组腺病毒载体p Adxsi-GFP-VP3,经酶切与测序鉴定证实连接正确,病毒滴度可达1.6×109 PFU/ml。RT-PCR显示转染48 h后Apoptin的m RNA表达;Western blot证实转染72 h后Apoptin蛋白获得高表达。TEM观察发现NCI-H460细胞凋亡早期染色质聚集,线粒体固缩,凋亡中晚期出现凋亡小体等超微形态学改变。MTT法检测显示转染后的NCI-H460细胞增殖活性明显降低,呈时间依赖性(F=93.384,P=0.000),与空病毒组和细胞对照组比较,差异有统计学意义(F=18.427,P=0.003)。FCM法检测显示Apoptin能有效地诱导NCI-H460细胞发生凋亡(F=47.307,P=0.000),且凋亡率随时间呈逐渐上升趋势(F=303.237,P=0.000);细胞周期表现为S期细胞比例降低(F=80.240,P=0.000),G2/M期细胞比例增高(F=110.080,P=0.000)。结论:Apoptin可以有效地诱导人大细胞肺癌NCI-H460细胞发生凋亡,这为Apoptin临床应用于人非小细胞肺癌基因治疗的深入研究奠定实验基础。

Objective：To construct recombinant adenovirus vector（p Adxsi-GFP-VP3）containing VP3 gene and to investigate the apoptosis-inducing effect of VP3 gene on human large cell lung cancer NCI-H460 cells. Methods：VP3 gene was gained by enzyme digestion with pc DNA3.0-VP3 recombined plasmid as a template and was diverted to shuttle vector. The p Adxsi-GFP-VP3 was obtained by connecting with p Adxsi vector. After enzymatic digestion and verification by sequencing,virus amplification and purification were conducted,and the titer of the recombinant adenovirus was determined by biological and physical assays finally. The p Adxsi-GFPVP3 was used to transfect human large cell lung cancer NCI-H460 cells with optimum multiplicity of infection. Expression of Apoptin in large cell lung cancer cells was respectively detected by reverse transcription polymerase chain reaction（RT-PCR）and Western blot. Cell ultrastructure at different periods of time after the transfection was observed by transmission electron microscopy（TEM）. Effect of Apoptin on cell proliferative vitality was determined by MTT assay. Apoptotic rate and cell cycle in NCI-H460 cells after transfection were measured by flow cytometry（FCM）. Results：The p Adxsi-GFP-VP3 was successfully constructed by enzyme digestion and sequencing identification and the virus titer was 1.6 ×109PFU/ml. RT-PCR results showed the m RNA expression of Apoptin in each experimental group at 48 h after the transfection. Western blot confirmed that Apoptin protein was highly expressed in each experimental group at 72 h after the transfection. The typical morphological changes in early apoptosis such as cell shrinkage,chromatin condensation and apoptotic bodies were observed under TEM. MTT assay indicated that the proliferative activity of transfected NCI-H460 cells was remarkably decreased and presented in a time-dependent manner（F=93.384,P=0.000）and there were statistical significances among different groups（F=18.427,P=0.003）. FCM detection showed that the expression of Apoptin in NCI-H460 cells after the transfection could induce cell apoptosis with a gradual increase in the apoptotic rate over time（F=47.307,P=0.000;F=303.237,P=0.000）. Moreover,the proliferation of NCI-H460 cells was slowed down;the proportion of cells was decreased at S phase（F=80.240,P=0.000）and was blocked at G2/M phase after the transfection compared with that of control group（F=110.080,P=0.000）. Conclusion：Our data demonstrate that Apoptin can effectively induce apoptosis of NCI-H460 cells,which may lay an experimental basis for applying Apoptin in human NSCLC gene therapy.