CAJ | 학술논문

目的探讨脆性组氨酸三联体基因(FHIT)转染对结肠癌细胞株SW480增殖和凋亡的影响及其作用机制.方法将重组真核表达质粒pRc/CMV2-FHIT通过脂质体转染技术导入人结肠癌细胞株SW480(实验组),筛选稳定转染的细胞并扩增培养,以转染了空质粒pRc/CMV2的SW480细胞作为阴性对照,以正常SW480细胞作为空白对照.应用MTT法检测细胞的增殖活性,流式细胞仪检测细胞周期分布和细胞凋亡率,Western blot分析caspase-8酶原的变化,半定量RT-PCR检测caspase-8 mRNA水平的改变,肽核酸标记底物的比色法检测caspase-8的相对活性.结果转染96 h后,实验组和阴性对照组细胞生长抑制率分别为71.7%和16.9%,G0/G1细胞比例分别为(63.3±3.5)%和(50.6±2.1)%,细胞凋亡率分别为(40.5±3.1)%和(18.6±2.6)%,caspase-8 mRNA条带光密度积分值分别为107和41,caspase-8蛋白相对活性分别为0.43和0.25;上述差异均有统计学意义(P＜0.05).当加入FHIT抑制剂后,caspase-8蛋白相对活性恢复至对照组水平(0.22).结论 FHIT基因转染能够明显抑制人结肠癌细胞株SW480的增殖,诱导SW480细胞发生G0/G1期阻滞,其作用机制可能与caspase-8表达及活性上调有关.
목적 탐토취성조안산삼련체기인(FHIT)전염대결장암세포주SW480증식화조망적영향급기작용궤제.방법 장중조진핵표체질립pRc/CMV2-FHIT통과지질체전염기술도입인결장암세포주SW480(실험조),사선은정전염적세포병확증배양,이전염료공질립pRc/CMV2적SW480세포작위음성대조,이정상SW480세포작위공백대조.응용MTT법검측세포적증식활성,류식세포의검측세포주기분포화세포조망솔,Western blot분석caspase-8매원적변화,반정량RT-PCR검측caspase-8 mRNA수평적개변,태핵산표기저물적비색법검측caspase-8적상대활성.결과 전염96 h후,실험조화음성대조조세포생장억제솔분별위71.7%화16.9%,G0/G1세포비례분별위(63.3±3.5)%화(50.6±2.1)%,세포조망솔분별위(40.5±3.1)%화(18.6±2.6)%,caspase-8 mRNA조대광밀도적분치분별위107화41,caspase-8단백상대활성분별위0.43화0.25;상술차이균유통계학의의(P＜0.05).당가입FHIT억제제후,caspase-8단백상대활성회복지대조조수평(0.22).결론 FHIT기인전염능구명현억제인결장암세포주SW480적증식,유도SW480세포발생G0/G1기조체,기작용궤제가능여caspase-8표체급활성상조유관.
Objective To investigate the effect of fragile histidine triad (FHIT) gene transfection on human colorectal cancer cell line SW480 through up-regulation of caspase-8 expression. Methods The eukaryotic expression plasmid containing FHIT,pRc/CMV2-FHIT was prepared and purified,and then identified by restrictive enzyme digestion. pRc/CMV2-FHIT was transfected into SW480 cells, and positive cell clones (SW480-FHIT, study group) were selected and amplified. Empty plasmid-transfected SW480 cells(SW480-pRc/CMV2, negative control) and normal SW480 cells (bland control) were used as control. Methyl thiazolyl tetrazolium (MTT) assay was used to test the changes in the proliferation of SW480 cells. Cell-cycle kinetics and apoptosis were analyzed by flow cytometry (FCM). The changes of pro-caspase-8, caspase-8 mRNA and caspase-8 relative activity were analyzed by Western blot, semi-quantitative RT-PCR and colorimetric assay with pan labeled substrate, respectively. Results At 96 hours after transfection, cell inhibition rates of the study group and the negative control group were 71.7% and 16.9%. G0/G1 ratio was(63.2±3.5)% and(50.6±2.1)%, optical density of caspase-8 mRNA band 107 and 41, and relative activity of caspase-8 0.43 and 0.25, respectively. All the differences above were statistically significant(P＜0.05). When FHIT inhibitor was added, the relative activity of caspase-8 decreased to 0.22, comparable to that in the control group. Conclusions FHIT gene transfection can significantly inhibit the proliferation and induce G0/G1 arrest in human colon cancer cell line SW480. The mechanism is related to the up-regulation of caspase-8 expression.