EB病毒潜伏膜蛋白1对人支气管上皮细胞转化作用机理的研究

背景与目的：目前许多研究显示潜伏膜蛋白1(latent membrane proteinl,LMPl)在一些上皮源性肿瘤的发生中可能起重要作用,但有关LMP1对正常上皮细胞作用的研究并不是很多。因而,本研究拟观察EBV-LMP1对人支气管上皮细胞(human bronchial epithelial,HBE)的转化作用,并初步探讨其作用机制。方法：将LMP1单独或与hTERT逆病毒表达载体共同转染HBE细胞,经抗性药物筛选获得阳性细胞克隆;通过绘制细胞生长曲线,进行软琼脂集落形成试验、端粒酶活性检测和cyclin A、p2l、CDK4蛋白表达的检测,研究LMPl对HBE细胞生物学特性的影响。结果：共同转染LMP1和hTERT的HBE细胞生长旺盛。各组细胞HBE／LMP1 hTERT、HBE／LMP1和HBE／pLNsx pBabe的软琼脂集落形成率分别为22．98％、16．94％和8．85％;端粒酶活性分别为2．825、2．44l、1．876。我们进行Western blot检测发现前两组细胞cyclin A表达上调、p2l表达下调,且两组之间无明显差异;而CDK4蛋白在各组之间表达一致。结论：在转化HBE细胞的过程中,LMP1和hTERT可能具有协同作用,通过调节cyclin A和p2l蛋白的表达水平,引起细胞异常增殖,使细胞向恶性化转变。

Transformation function of human bronchial epithelial cell by latent membrane protein1 encoded by Epstein-Barr virus gene

BACKGROUND &OBJECTIVE:Althoug h some results show that latent membrane protein 1(LMP1)may play an important role in the development of epithelium-derived tumors.There are few studies on the relationship between LMP1and norma l epithelial cell.So this study was desig ned to evaluate the effect of EB V LMP1on cell transformation in huma n bronchial epithelial (HBE)cell and its mechanism.METHODS :Retrovirus expression vectors(LMP1and LMP1+hTERT)were transfected into HBE cells and selected with the appropriate dr ug s to g et resistant cells.The biolo g ical characteristics were studied by cel l g rowth curve,colony formation analysis,and the determination of telomerase act ivity and protein expression of Cycl in A,p21,and CDK4.RESULTS:wt-LMP1tog ether with hTERT g ene transfected cells g r ew rapidly than HBE /wt-LMP1cells.Sof t ag ar plating rates of HBE /wt-LMP1-hTERT,HBE /wt-LMP1,and HBE /pLNSX+pBabe were 22.98%,16.94%,and 8.85%,respectively.Th e activities of telomerase were 2.825,2.441,and 1.876,respectively.In a ddition,there was no difference of protein expression of CDK4among these g roup s.However,HBE /wt-LMP1-hTERT and HBE /wt-LMP1cells had low-level p21and hig h-level cyclin A protein expr ession than the vector g roups.CONCLUSION:The results sug g est that LMP1is in tandem with hTERT in promoting cell p roliferation and transformation th roug h modulating expression level of cycl in A and p21protein.