EB病毒潜伏性膜蛋白1促原代MEF细胞永生化的作用及机制

为了探讨EB病毒潜伏性膜蛋白1(LMP1)促原代小鼠胚胎成纤维(MEF)细胞增殖规律及作用机制,构建包含野生型LMP1和其TNFR相关死亡区(TRADD)结合区即羧基末端384～386氨基酸置换(YYD→ID)的突变型LMP1逆转录病毒,感染原代培养的MEF细胞,动态观察各细胞在传代培养过程中细胞动力学和形态学变化。发现对照细胞(MEF-LNSX)传至P8～P10时出现明显的生长阻滞;携突变型LMP1的细胞(MEF-LMP1TRADD)自P10起倍增时间逐渐延长,P19时出现明显生长阻滞;而携野生型LMP的细胞(MEF-LMP1)倍增时间逐渐降低并发生了永生化。Westernblot检测发现MEF-LNSX细胞CyclinA表达自P4起明显降低,MEF-LMP1TRADD细胞自P10显著增高后快速降低,MEF-LMP1自P10显著增高后一直维持在较高水平。结果表明:LMP1能促进MEF细胞增殖并诱导体外永生;LMPTRADD则仅能诱导MEF细胞的早期增殖。提示羧基末端区(TRADD)是LMP1促MEF细胞永生的重要活性部位,上调CyclinA表达可能是其作用机制之一。

Action and Mechanism of Epstein Barr virus Latent Membrane Proteinl induced Immortalization of Mouse Embryonic Fibroblasts

To investigate the action and mechanisms of Epstein Barr-virus(EBV)latent membrane protein1(LMP1)in stimulating the proliferation of primary mouse embryonic fibroblast(MEF)cells the retrovirus(RV)containing wild type LMP1(wt-LMP1)and mutant type LMP1(mt-LMP1),which replaced YYD with ID in tumor necrosis factor receptor associated death domain(TRADD)-binding site,were constructed and used to infect the MEF cells,respectively.Then cytokinetic and morphologic changes from infected cells at the course of passage were observed and found that control cells(MEF-LNSX)showed an apparent growth arrest from passages 8(P8).In contrast,mt-LMP1 prolonged the cell(MEF-LMP1TRADD)doubling time since P10 and arrested growth at P19,while the MEF-LMP1 cell with wt-LMP1 had an accurate doubling time and displayed a immortalization.Meantime,the expression of cyclinA had a decrease in MEF-LNSX and MEF-LMP1TRADD after P4and P10 respectively.while in MEF-LMP1,cyclinA increased from P10 and kept at a high level.These results suggested that LMP1 can stimulate proliferation of MEF cells and induce their immortalization,while LMP1TRADD only induces an early proliferation of MEF cells.It is implied that TRADD domain might involve in LMP1-induced MEF cells immortalization.