摘要
目的探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)对膀胱癌细胞的抑制作用。方法构建TRAIL融合增强绿色荧光蛋白(EGFP)真核表达载体,脂质体法转染膀胱癌细胞株EJ细胞,荧光显微镜下计算转染率。分别采用逆转录-聚合酶链反应(RT-PCR)、Western blot检测 TRAIL的表达。采用流式细胞仪检测细胞凋亡,噻唑蓝(MTT)法、细胞倍增时间和集落形成率观察TRAIL的抑制作用。结果 48 h观察重组载体转染率为38．4％,空载体转染率为35．3％(P> 0．05),RT-PCR和Western blot证实转染后EJ细胞中TRAIL的表达明显增加;凋亡率明显高于转染空白载体和未转染组(P<0．01);EJ细胞增殖受到明显抑制(P<0．01)。结论 TRAIL具有诱导膀胱癌细胞凋亡、抑制增殖的作用,有望成为治疗膀胱癌的新方法。
Objective To investigate the inhibitory effects of tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） gene on the apoptosis and the growth of human bladder carcinoma cells. Methods The eDNA encoding full-length human TRAIL was cloned. The eukaryotic expression vector pEGFP-N1-TRAIL containing enhanced green fluorescent protein （EGFP） gene and expressing TRAIL was constructed, then transfected into EJ cells using a liposome-mediated transfection system. EGFP gene as a report gene was detected by fluorescent microscopy. TRAIL expression was detected by RT-PCR and Western blot. The apoptosis was determined by flow cytometry, and the cell growth inhibition were assessed by MTT, cell doubling generation time and colonyforming efficiency. Results Twenty-four h after transfection, fluorescence microscopy showed great number of EJ cells that expressed green fluorescence in both the pEGFP-N1 group and pEGFP-N1-TRAIL group. The rates of green fluorescent protein positive cells were 38.496 and 35.396 respectively in these two groups （P〉0.05）. RT-PCR and Western blot revealed TRAIL was increased significantly after transfection with TRAIL. A significantly higher rate of apoptosis was found in EJ cells transfected with TRAIL （P〈0.01）.The proliferation of the ceils transfected with TRAIL was significantly decreased （P 〈 0.01）. Conclusion TRAIL may induce apoptosis and inhibit the proliferation of human bladder carcinoma cells, and may become a novel approach to treatment of cancer.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2006年第5期568-570,共3页
Chinese Journal of Experimental Surgery
