摘要
目的:探讨sTRAIL联合融合受体eDR4/iFas对HT-29细胞的凋亡作用。方法:采用PCR方法分别扩增eDR4和iFas,通过重叠PCR将两者连接起来组成融合受体基因eDR4/iFas,将其克隆到带Survivin启动子的表达载体上,另外采用组成型启动子CAG调控sTRAIL表达。体外转染结直肠癌细胞株HT-29和人胚肾细胞HEK293,MTT法检测两种细胞的存活率,RTPCR和Western Blot检测目的蛋白的表达,Hoechst染色进一步分析eDR4/iFas联合sTRAIL对HT-29细胞的凋亡效果。结果:在HT-29细胞中,sTRAIL组的细胞存活率为40.9%±18.7%,而eDR4/iFas+sTRAIL组的细胞存活率为21.6%±9.1%,两者间存在统计学差异(P<0.05)。结论:eDR4/iFas能提高sTRAIL对HT-29细胞凋亡的敏感性,而对正常细胞HEK293没有毒副作用。
ABSTRACT AIM： To investigate the effect of sTRAIL combined with infusion receptor eDR4/ iFas on apoptosis of HT-29 cells. METHODS： In this study, eDR4 and iFas genes were amplified respectively by PCR and used to generate fusion gene named eDR4/iFas by splicing overlap extension PCR. eDR4/iFas was cloned into the expression vector with the tumor-specific survivin promoter, and expressed sTRAIL with constitutive promoter CAG. After treatment with eDR4/iFas and sTRAIL, the cell viability analysis of both colorectal cancer cells HT-29 and human embryonic kidney cells HEK293 was assayed by MTT. Both TRAIL mRNA and protein expressions were detected with RT-PCR and ELISA respectively. The apoptosis of HT-29 cells was analyzed by Hoechst staining. RESULTS. In HT-29 cells, cell survival rate in sTRAIL group was 40.9%±18.70%, while cell survi, val rate in eDR4/iFas＋sTRAIL group was 21.6%±9.1%, there was significant difference between the two groups （P〈0.05）. CONCLUSION： The results have revealed that eDR4/iFas can improve TRAIL-induced apoptosis of HT-29 cells, has no toxicity for HEK293 cells.
出处
《中国临床药理学与治疗学》
CAS
CSCD
2014年第9期966-972,共7页
Chinese Journal of Clinical Pharmacology and Therapeutics
基金
中央高校基本科研业务费资助项目(JB-ZR1142)
福建省自然科学基金(2010J01207)资助
