人端粒酶反转录酶催化酶亚单位基因对肝母细胞瘤细胞株体外生物学特性的影响  

Effects of antisense human telomerase reverse-transcript protein subunit (hTERT)gene on biological characteristics of hepatoblastoma cell line in vitro

作　　者：刘磊[1] 李成荣[1] 孙来保[1] 王国兵[1] 王兵[1] 

机构地区：[1]深圳市儿童医院外科,518026

出　　处：《中华儿科杂志》2004年第7期481-485,共5页Chinese Journal of Pediatrics

摘　　要：目的　研究人端粒酶反转录酶催化酶亚单位 (hTERT)基因对肝母细胞瘤细胞株体外生物学特性的影响。方法　体外将已构建好的正、反义hTERT真核表达载体转染HepG2 肝母细胞瘤细胞株 ,经G4 18及聚合酶链反应 (PCR)筛选鉴定获得分别转入了正、反义hTERT载体的抗性克隆细胞HepG2 s和HepG2 as。运用逆转录 PCR(RT PCR)技术及TRAP 银染法对各组细胞内源性hTERTmRNA的表达及端粒酶的活性进行了检测 ;同时还采用MTT法、双层软琼脂克隆形成试验、流式细胞术观察和分析了反义hTERT对肝母细胞瘤细胞体外生长增殖活力的影响及是否能诱导瘤细胞的凋亡。结果　与空白对照组、正义hTERT组相比 ,反义hTERT能显著降低HepG2 细胞内源性hTERTmRNA的表达 (n =10 ,t =7 6 1,P <0 0 1)和下调端粒酶活性。当各组细胞传至第 2 0代后 ,与HepG2 、HepG2 s比较 ,HepG2 as细胞的生长速度和集落形成能力明显地减慢和降低 (n =10 ,t=4 5 4 ,P <0 0 1和n =10 ,t=3 96 ,P <0 0 1) ,同时伴有凋亡率的显著增加 (n =10 ,t=9 2 4 ,P <0 0 1和n =10 ,t=8 37,P <0 0 1)。结论　反义hTERT体外可抑制肝母细胞瘤细胞的生长增殖能力 。Objective Telomerase, a complex of ribose and nucleoprotein, is a specific marker of tumor, which expresses in 98% infinite cell lines and 90% malignant tumor organizations and whose function is to maintain the length of telomere. Human telomerase reverse transcript protein subunit(hTERT)is the key element and rate limiting factor of elomerase activity. Our study was to investigate the effects of antisense hTERT gene on biological characteristics of hepatoblastoma cell line in vitro .Methods The sense and antisense hTERT eukaryotic expression vectors that we had constructed before were transfected into hepatoblastoma cell line HepG 2 by using the SuperFect transfection reagent (Qiagen) according to the manufacturer′s instructions, then the HepG 2 s and HepG 2 as of G418 resistant colonies were obtained with G418 and identified for the presence of hTERT insert by PCR with T7 and pcDNA3 1/BGH reverse primers. After that, we have detected the endogenous hTERT mRNA expression and telomerase activity by quantitative real time RT PCR and TRAP silver staining assay in cells from each group. Meanwhile, MTT cellular proliferation assay, soft agar colony formation assay and flow cytometry were employed to analyze if the proliferation capacity of liver cancer cells was affected in vitro and the tumor cells could be induced to apoptosis by antisense hTERT.Results Antisense hTERT significantly down regulated the endogenous hTERT mRNA expression (15 35±1 72/HepG 2 as, 43 8±2 89/ HepG 2 s,45 2±3 46/HepG 2)( n =10, t =7 61, P <0 01) and telomerase activity in HepG 2, compared to blank control and sense hTERT. After 20 passages of three group cells, a 7 day cell growth curve and the numbers (size) of soft agar colony formation showed the proliferation and the anchorage independent growth in HepG 2 as were significantly suppressed(50 6±4 8/HepG 2 as,113 52±8 15/HepG 2 s, 119 12±10 82/HepG 2)( n =10, t =4 54, P < 0 01 and n =10, t =3 96, P <

关 键 词：人端粒酶反转录酶催化酶亚单位基因 肝母细胞瘤 肿瘤细胞 生物学特性 端粒酶活性 

分 类 号：R735.7[医药卫生—肿瘤]