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

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作者单位:518026深圳市儿童医院外科(E 2mail :liulei3322@ )

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

刘磊　李成荣　孙来保　王国兵　王兵

【摘要】　目的　研究人端粒酶反转录酶催化酶亚单位(h TERT )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h TERT 真核表达载体转染Hep G 2肝母细胞瘤细胞株,经G 418及聚合酶链反应(PCR )筛选鉴定获得分别转入了正、反义h TERT 载体的抗性克隆细胞Hep G 22s 和Hep G 22as 。运用逆转录2PCR (RT 2PCR )技术及TRAP 2银染法对各组细胞内源性

h TERT mRNA 的表达及端粒酶的活性进行了检测;同时还采用M TT 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h TERT 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h TERT 组相比,反义h TERT 能显著降低Hep G 2细胞内源性h TERT mRNA 的表达(n =10,t =7161,P <0101)和下调端粒酶活性。当各组细胞传至第20代后,与Hep G 2、Hep G 22s 比较,Hep G 22as 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

=4154,P <0101和n =10,t =3196,P <0101),同时伴有凋亡率的显著增加(n =10,t =9124,P <0101和n =10,t =8137,P <0101)。结论　反义h TERT 体外可抑制肝母细胞瘤细胞的生长增殖能

力,在抗肿瘤基因治疗中具有特异的靶向性和潜在应用的广谱性。

【关键词】　端粒,末端转移酶;　肝胚细胞瘤;　逆转录聚合酶链反应;　细胞分裂;　肿瘤细胞,培养的治疗

E ffects of antisense human telomerase reverse 2transcript protein subunit (hTERT)gene on biological characteristics of hepatoblastoma cell line in vitro L IU L ei ,L I Chen 2rong ,S UN L ai 2bao ,W A N G Guo 2bing ,W A N G Bing.Depart ment of S urgery ,S henz hen Children ′s Hospital ,S henz hen ,518026China

【Abstract 】　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 2transcript protein subunit (h TERT )is the key element and rate 2limiting factor of elomerase activity.Our study was to investigate the effects of antisense h TERT gene on biological characteristics of hepatoblastoma cell line in vit ro .Methods The sense and antisense h TERT eukaryotic expression vectors that we had constructed before were transfected into hepatoblastoma cell line Hep G 2by using the SuperFect transfection reagent (Qiagen )according to the manufacturer ′s instructions ,then the He p G 22s and Hep G 22as of G 4182resistant colonies were obtained with G 418and identified for the presence of h TERT insert by PCR with T7and pcDNA311/B GH reverse primers.After that ,we have detected the endogenous h TERT mRNA expression and telomerase activity by quantitative real 2time RT 2PCR and TRAP 2silver staining assay in cells from each group.Meanwhile ,M TT 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 vit ro and the tumor cells could be induced to a poptosis by antisense h TERT.R esults Antisense h TERT significantly down 2regulated the endogenous h TERT mRNA expression (15135±1172/Hep G 22as ,4318±2189/Hep G 22s ,4512±3146/Hep G 2)(n =10,t =7161,P <0101)and telomerase activity in Hep G 2,compared to blank control and sense h TERT.After 20passages of three group cells ,a 72day cell growth curve and the numbers (size )of soft agar colony formation showed the proliferation and the anchorage 2independent growth in Hep G 22as were significantly suppressed (5016±418/Hep G 22as ,113152±8115/Hep G 22s ,119112±10182/Hep G 2)(n =10,t =4154,P <0101and n =10,t =3196,P <0101),compared to Hep G 2and Hep G 22s.However there was a significant increase in apoptosis percentage of Hep G 22as by flow cytometry