论文部分内容阅读
目的:构建了人端粒酶逆转录酶(humantelomeraseresersetranscription,hTERT)基因核心启动子调控的肿瘤坏死因子相关凋亡诱导配体(TNF-relatedapoptosisinducingligand,TRAIL)基因真核表达载体并观察其对卵巢癌细胞凋亡的诱导作用。方法:从人胎盘中提取总RNA,利用RT-PCR技术扩增了TRAIL基因片段。测序正确后,利用基因重组方法将TRAIL基因克隆入带有hTERT基因核心启动子pIRES2-EGFP真核表达载体中。获得由hTERT基因核心启动子调控的绿色荧光蛋白和带有效应型TRAIL基因的真核表达载体hTER-Tpromoter-pIRES2-EGFP-TRAIL。用脂质体转染法,将其转染卵巢癌细胞系SKOV3细胞中,应用RT-PCR法检测转染前后卵巢癌细胞中外源基因的表达情况,以流式细胞术检测TRAIL对卵巢癌细胞的细胞周期的影响及诱导凋亡的作用。结果:经酶切鉴定及测序结果证实,所构建的重组载体正确。转染hTERTpromoter-pIRES2-EGFP-TRAIL载体后,SKOV3细胞的增殖受到抑制,出现明显凋亡。结论:hTERT基因核心启动子在卵巢癌细胞中可特异性地调控其下游TRAIL基因的表达,并发挥其促凋亡作用。构建由hTERT基因核心启动子调控的表达载体,可能是一种新型和有希望的肿瘤治疗的途径。
OBJECTIVE: To construct an eukaryotic expression vector of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene regulated by human telomerase reverse transcriptase (hTERT) gene core promoter and observe its effect on ovarian cancer cells Apoptosis induction. Methods: Total RNA was extracted from human placenta and the TRAIL gene fragment was amplified by RT-PCR. After sequencing, the TRAIL gene was cloned into eukaryotic expression vector pIRES2-EGFP with hTERT gene core promoter by gene recombination method. The green fluorescent protein regulated by the hTERT gene core promoter and the eukaryotic expression vector hTER-Tpromoter-pIRES2-EGFP-TRAIL with the effector TRAIL gene were obtained. The transfected cells were transfected into ovarian cancer cell line SKOV3 by lipofectamine 2000. The expression of exogenous genes in ovarian cancer cells before and after transfection was detected by RT-PCR. The effect of TRAIL on ovarian cancer cells The cell cycle and the role of apoptosis induction. Results: The results of enzyme digestion and sequencing confirmed that the constructed recombinant vector was correct. After hTERT promoter-pIRES2-EGFP-TRAIL vector was transfected, the proliferation of SKOV3 cells was inhibited and obvious apoptosis occurred. CONCLUSION: The core promoter of hTERT gene can specifically regulate the expression of downstream TRAIL gene in ovarian cancer cells and exert its pro-apoptotic effect. Construction of an expression vector regulated by the core promoter of hTERT gene may be a new and promising approach to tumor therapy.