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1、乙型肝炎病毒X蛋白诱导肝卵圆细胞恶性转化廖凯,王月飞,卢乐乐,张雷达(400038,重庆,第三军医大学西南医院肝胆外科)[摘要]目的研究乙型肝炎病毒X蛋白(HepatitisBvirusX,HBx)对肝卵圆细胞的转化作用。方法构建表达HBx和嘌呤霉素抗性的慢病毒LV-HBx-PM及对照慢病毒LV-PM分别感染WB-F344肝卵圆细胞。以感染HBx慢病毒载体WB-F344细胞作为实验组(WB-F344-HBx),以感染对照慢病毒载体的WB-F344细胞作为阴性对照组(WB-F344-PM),以未转染慢病毒载体的培养相同代数的WB-F344细胞
2、作为空白对照组(WB-F344-Mock)。通过real-timeRT-PCR和Westernblot验证各组细胞中HBx的表达;运用MTT法、流式细胞术、细胞迁移实验,裸鼠移植瘤实验观察各组细胞的增殖能力、细胞周期、迁移能力和成瘤能力。结果WB-F344-HBx细胞在基因水平和蛋白水平稳定高效表达HBx;WB-F344-PM和WB-F344-Mock不表达HBx。与阴性对照组WB-F344-PM相比,实验组WB-F344-HBxG0/G1期细胞比例降低(55.98%±0.39%,61.65%±1.37%,P=0.003),G2/M期细胞比
3、例升高(15.05%±1.67%,7.83%±0.46%,P<0.001),细胞周期进程加快,细胞增殖能力增强(P<0.001),具有更强的细胞迁移能力(P<0.001)和成瘤能力;而阴性对照组WB-F344-PM与空白对照组WB-F344-Mock相比,细胞周期、细胞增殖能力、细胞迁移能力和成瘤能力无显著变化。结论乙型肝炎病毒X蛋白可诱导肝卵圆细胞恶性转化。[关键词]乙型肝炎病毒;X蛋白;卵圆细胞;恶性转化[中图分类号]R735.7[文献标志码]AHepatitisBvirusXproteininduceshepaticovalcellm
4、alignanttransformationLiaoKai,WangYuefei,LuLele,ZhangLeida(DepartmentofHepatobiliarySurgery,SouthwestHospital,ThirdMilitaryMedicalUniversity,Chongqing400038,China)[Abstract]ObjectiveTheaimofthisstudywastodetectthemalignanttransformationofhepatitisBvirusX(HBx)proteinonhepat
5、icovalcells.MethodsTheWB-F344cellinfectingwithlentivirusexpressingHBxandpuromycin(PM)-resistantgenewasdefinedasexperimentalgroup(WB-F344-HBx).TheWB-F344cellinfectingwithlentivirusonlyexpressingpuromycin-resistantgenewasdefinedasnegativecontrolgroup(WB-F344-PM).TheWB-F344cell
6、withoutinfectingwithlentiviruswasdefinedasblankcontrolgroup(WB-F344-Mock).TheexpressionsofHBxinthethreegroupswereconfirmedbyreal-timeRT-PCRandWesternblotanalysis.Thecellcycle,proliferation,abilityofmetastasisandtumorigenicityweredetectedwithflowcytometry,MTTassay,metastasis
7、experimentandnudemousetransplantationtumorexperiment,respectively.ResultsWB-F344-HBxhighlystableexpressedHBxproteinwhileWB-F344-PMandWB-F344-MockwithoutexpressingofHBx.ComparedwithWB-F344-PM,WB-F344-HBxstatecansignificantlyacceleratetheprocessofG1/Scellcycle,promotethecell
8、proliferation,andstrongerabilityofmetastasisandtumorigenicity.Therewasnosignificantdiffer