临床检验医学网-幸福检验

【摘要】  目的：构建转基因细胞株Bel7404/HBx,研究HBx对肝癌细胞周期及凋亡的影响，为探索HBx与原发性肝细胞癌（HCC）之间的关系提供更为完善的理论依据。方法：运用脂质体转染及G418筛选获取Bel7404/HBx阳性克隆，并分别用聚合酶链反应（RTPCR）和Westernblot鉴定HBx mRNA与蛋白的表达，进一步用四唑兰比色实验（MTT）检测Bel7404/HBx的增殖，流式细胞仪（FCM）检测Bel7404/HBx的细胞周期及凋亡。结果：成功构建Bel7404/HBx，MTT检测结果表明Bel7404 /HBx生长速度加快，流式细胞仪检测结果显示,与对照组细胞相比, Bel7404 /HBx凋亡率明显降低（P<0.05)，G1期细胞比例降低（P<0.05), S期及G2期细胞所占比例增高（P<0.05)。结论:HBx能加速细胞周期进程，从而促进肝癌细胞的生长并抑制细胞凋亡，可能对肝癌细胞恶性程度具有重要的影响。

【关键词】  HBx；原发性肝细胞癌；聚合酶链反应；免疫印迹；四唑兰比色实验；流式细胞术

［中图分类号］ R373.2;R512.62  ［文献标识码］ A  ［文章编号］ 10071237(2010)09113504

 作者：林松挺1 ,陈正义1，陈莹晖2 ,程 斌3    作者单位：海南医学院科研基金资助学报项目（0020100315）(1 中南大学湘雅医学院附属海口医院消化内科，海南 海口 570208；2 海南医学院附属医学中心ICU，海南 海口 570102；3 华中科技大学同济医学院附属同济医院，湖北 武汉 430030）

Effect of X gene of gepatitis B virus on cell cycles of hepatoma cells Bel7404

 LIN Song ting1 ,CHEN Zhengyi1,CHEN Yinghui2,CHENG Bin3

（1 Department of Gastroenterology, Affiliated Haikou Hospital of Xiangya Medical Univercity, Central South College, Haikou 570208；2 ICU of Medical Center, Affiliated Hospital of Hainan Medical University，Haikou 570102；3 Affiliated Tongji Hospital of Huazhong University of Science and Technology, Tongji Medical College,Wuhan 430030, China）

［Foundation Project］: Hainan Medical University Scientific Research Fund Supported Project of the Journal(0020100315)

［Author］:LIN Songting (1973), Male, Dongfang Hainan, M.M., Tel: 13398901463, Email：linst9999@126.com.

Received: 20100719    Revised: 20100812 JHMC,2010;16(9):11351138

   
View from specialist: It is creative, and of certain scientific and educational value.

［ABSTRACT］ Objective: To establish transgenic cell lines Bel7404/HBx, study the influence of HBx on cell cycles and apoptosis, and provide more extensive theories for exploring the relationship between HBx and hepatocellular carcinoma (HCC). Methods: The Bel7404/HBx positive clones were obtained by Lipofectamine tranfection and G418 selection, then HBx mRNA and protein were detected by reverse transcription polymerase chain reaction(RTPCR) and Western blot, respectively. The tetrazolium blue colorimetric test (MTT) and flow cytometer ( FCM) were used to measure proliferation, cell cycles and apoptosis of Bel 7404/HBx. Results: Bel7404/HBx was successfully established. MTT showed that the Bel7404 / HBx grew faster than the control group （P<0.05), and FCM showed that compared with cells in control group, the apotosis rates of Bel7404/HBx were at a low level, cells in G1 phase decreased but increased in S and G2 phase in proportion（P<0.05). Conclusions: HBx can accelerate cell cycle progression to promote the growth of hepatoma cells but inhibit apoptosis, which indicates that it may have great influence on the malignant degree of hepatocellular carcinoma.

   
［KEY WORDS］ HBx; Hepatocellular carcinoma; RTPCR; Western blot; MTT; FCM