摘要
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.
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.
出处
《中华胃肠外科杂志》
CAS
北大核心
2010年第9期691-694,共4页
Chinese Journal of Gastrointestinal Surgery
基金
广东省自然科学基金（06020005）
