摘要
目的:探讨Apoptin基因对人胃癌细胞BGC-823的抑制作用及其参与胞内细胞信号转导的机制.方法:应用脂质体转染法将重组质粒pVAX1-Apoptin体外转染BGC-823细胞,运用噻唑兰(methylthiazolyltetrazolium,MTT)分析法检测重组质粒对BGC-823肿瘤细胞的抑制作用;通过AO/EB染色法对pVAX1-Apoptin转染的肿瘤细胞进行形态学观察;运用流式细胞术检测细胞线粒体跨膜电位(△Ψm);通过免疫印迹检测细胞色素c(cytochromec,Cytoc)释放;应用底物显色法检测Caspase-3/9活性.结果:pVAX1-Apoptin转染比pVAX1具有更强的抑瘤效应(10μg质粒转染72h后,23.37%vs99.61%,P<0.01),并且肿瘤细胞的死亡方式以凋亡为主.实验结果显示,pVAX1-Apoptin转染导致细胞线粒体跨膜电位下降,与pVAX1转染BGC-823细胞相比有显著差异(46.7%±7.26%vs70.66%±5.98%,P<0.01);同时伴随细胞色素c的释放.另外,pVAX1-Apoptin和pVAX1转染细胞的Caspase-3/9活性具有显著差异(Caspase-9:0.181±0.032vs0.079±0.013,P<0.01;Caspase-3:0.242±0.041vs0.058±0.023,P<0.01).结论:pVAX1-Apoptin转染BGC-823肿瘤细胞导致线粒体跨膜电位下降,并刺激释放细胞色素c,从而激活Caspase-9.经细胞色素c激活的Caspase-9进而活化凋亡通路下游关键作用因子,并对BGC-823肿瘤细胞产生抑制效应.
AIM： To investigate the anti-tumor effects of Apo- ptin gene on human gastric carcinoma BGC-823, and to explore the mechanism of apoptin in the participation of signal transduc-tion pathway.
METHODS： Recombinant plasmid pVAX1- Apoptin was transfected into BGC-823 cells by application of liposome in vitro. The anti-tumor effect on BGC-823 cells was measured through methyl thiazolyl tetrazolium （MTT） assay, and the morphological changes of pVAX1-Apoptin- transfected cells were observed by AO/EB staining. Furthermore, the rnitochondrial trans- membrane potential （AWm） was analyzed by flow cytometry and the release of cytochrome c （Cyto c） was detected by Western blot assay. Colorimetric assay was used to analyze the activities of Caspase-3/9.
RESULTS： pVAX1-Apoptin caused a more powerful cytotoxic effect than pVAX1 did （72 h after 10μg plasmid transfection, 23.37% vs 99.61%, P 〈 0.01）, and apoptosis was a dominant death style in the pVAX1-Apoptin-transfected cells. Furthermore, in pVAX1-Apoptin-transfected cells, an increase of cytosolic cytochrome c content co-occurred with a loss of trans-membrane potential （compared with pVAXl-transfected BGC-823 cells, 46.7% ± 7.26% vs 70.66% ± 5.98%, P 〈 0.01）. In addition, a significant difference was found between pVAX1-Apoptin- and pVAXl-transfected cells in Caspase-9 and Cas- pase-3 activities （Caspase-9：0.181 ± 0.032 vs 0.079±0.013, P 〈 0.01; Caspase-3：0.242 ± 0.041 vs 0.058 ±0.023, P 〈 0.01）.
CONCLUSION： In BGC-823 cells, pVAX1- Apoptin induced collapse of the mitochondrial trans-membrane potential can exert a feedback effect to elicit cytochrome c release, and then lead to the activation. Cytochrome c-dependent Caspase-9 activation stimulates the key down- stream factors of apoptotic pathway, exhibiting an inhibitory effect on BGC-823 cells.
出处
《世界华人消化杂志》
CAS
北大核心
2006年第31期2991-2996,共6页
World Chinese Journal of Digestology
基金
国家自然科学基金重大项目资助项目
No.39893290-4-3~~
