PAR-2基因在EBV转化人胃上皮细胞中的作用
刘换新 陈娟 刘梅 叶春
【摘要】 目的研究EBV感染人胃上皮细胞系GFS-1后PAR-2基因的表达状况及转化作用，以探讨PAR-2基因在Epstein-Baar病毒（EBV）相关胃癌发生中所起的作用。方法用携带NEO基因的重组EBV产生细胞系Akata 1061以密切接触法感染人胃上皮细胞系GF5-1,采用脂质体介导法将PAR-2基因转染至同一细胞，以pcDNA3空载体质粒转染同一细胞为对照；经G418筛选获得感染或转染的抗性细胞克隆；采用免疫细胞化学法测定EBNA1的表达以鉴定EBV感染细胞克隆，测定EBV感染和PAR-2基因转染细胞中PAR-2蛋白的表达；通过形态学观察、细胞生长曲线测定及流式细胞分析等方法观察细胞生物学特性的变化。结果与对照组相比，EBV感染及PAR-2转染后细胞发生明显的形态学变化，生长速度明显加快，S期细胞比例显著提高，分别为（70.35±0.91）%、（60.67±3.06） % 和（34.74±1.03）%,差异有显著性（P<0.05），表明EBV感染及PAR-2基因转染均使细胞增殖加快。结论EBV感染及PAR-2基因转染可使人胃上皮细胞的增殖速度加快，细胞恶性程度增强，但并未导致肿瘤的发生。EBV对人胃上皮细胞的转化作用可能与PAR-2基因的过度表达有关。
【关键词】 Epstein-Barr 病毒（EBV）；PAR-2;人胃上皮细胞；基因转染；细胞周期
Role of PAR-2 gene in EBV-transformed human gastric epititelial cells
LIU Huan-xin,CHEN Juan,LIU Mei,et al.The Hospital of Armed Police of Guangdong,Guangzhou 510507,China
[Abstract]ObjectiveTo define the role of PAR-2 gene in the carcinogenesis of EBV-associated gastric carcinoma, we examined the expression of PAR-2 in the human epithelial cell line GES1 infected with EBV and tested if EBV could induce cell transformation.MethodsAkata 1061 was used to infect GES-1 the intimate contact meth-od. pcDNA3-PAR-2 was hansfected into cells the UpofectAMINE method, whereas pcDNA3 vector was used as the control. Geneticin 418 （G418） was adopted to select positive clones that were either EBV or PAR-2 positive. Immuno-histochemical staining method was used to detect EBNAI expression in cell clones infected with EBV and the expressionof PAR-2 protein in EBV-infected or PAR-2-transfected cell clones. Morphological observations, cell growth curve de-terminations, soft agar gel formation tests, and flow cytometry were employed to measure changes in cell proliferationand cell cycle progress.ResultsCompared to the control, EBV-infected or PAR-2-transfected cells revealed significant morphological changes that were accompanied a significant increase in the cell growth velocity. The numbers of S ase cells were significantly increased（P<0.05）， both EBV-infection （70.35±0.91%）and PAR-2-transfection（60.67%±3.06%），compared to the control （34.74%±1.03%）。Cell colony formation was not detected the soft agar gel clone formation test.ConclusionBoth EBV infection and PAR-2 transfection promote the proliferation of gastric epithelial cells result in an increased tendency of malignance. However, the both events failed to induce carcinoma. These data suggest that an over-expression of PAR-2 may play a role in EBV induced transformation on human gastric epithelial cells.
[Key words]Epstein-Barr virus（EBV ）； PAR-2; Human gastric epithelial cell; Gene transfection; Cell cycle
胃癌是我国最常见的消化道恶性肿瘤，发病率在世界范围内的恶性肿瘤中占第二位，其发病机制目前并不十分清楚。大多数学者认为，胃癌的发生是物理、化学、生物等多因素、多阶段的发展过程。1990年Burke等[1]首次报道了1例EBV阳性胃癌，自此EBV感染与胃癌的关系受到关注[2].EBV最初是由Epstein和Barr从非洲伯基特淋巴瘤（BL）培养中发现的，它与多种人类肿瘤有关。目前，对EBV相关胃癌的研究主要在临床病理方面，而EBV感染后胃上皮细胞变化的分子机制尚不明确。笔者采用EBV感染和PAR-2基因转染体外培养的胃上皮细胞系GES-1,对比分析细胞生物学特性的变化，从而探讨PAR-2基因在EBV致胃癌发生发展中的作用。
1材料与方法
1.1主要试剂脂质体Lipofect AMINETM 2000 regent,G418购自Invitrogen公司；小量质粒提取试剂盒、限制性内切酶、XDNA/Hind m maker,200 by DNA stepladder、琼脂糖购自华美生物技术工程公司；RPMI1640培养基购自美国Gibco公司；胎牛血清（FCS）购自基因公司；免疫细胞化学染色试剂盒购自北京中杉金桥生物技术有限公司；胃癌病人血清为来自本院肿瘤科；载体和细胞株：pcDNA3空载体质粒、pcDNA3-c-myC重组质粒及携带NEW基因的重组EBV产生细胞系Akata 1061均为广州安必平生物科技有限公司提供；人胃上皮细胞系GFS-1由南方医科大学肿瘤研究所提供；大肠杆菌HB 101由安必平生物科技有限公司提供。
1.2细胞的培养人胃上皮细胞系GES-1用含10%FCS,100 U/ml青霉素、100 U/ml链霉素的RPMI 1640培养基，置于
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