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为了探讨EB病毒编码的潜伏膜蛋白 1(EBV LMP1)促进细胞增殖 ,参与EBV相关疾病致瘤的分子机制 ,研究了LMP1在鼻咽癌细胞中调节cyclinD1表达 ,进而影响细胞周期行进及细胞恶性表型改变 ,并初步确定了LMP1发挥该功能的结构域 .利用已建株的Tet on LMP1 HNE2鼻咽癌细胞系 ,蛋白质印迹实验分析LMP1诱导cyclinD1蛋白质表达的表达动力学 ,包括时间效应及剂量效应 ;利用三种LMP1功能区缺失的突变体及野生型LMP1,以载体型细胞为对照 ,确定LMP1活化cyclinD1表达的结构域 .同时结合基因诱导表达及反义寡聚核酸技术阻断基因表达的实验方法 ,进一步确定LMP1上调的cyclinD1功能 ,即对细胞周期行进及细胞恶性表型的影响 .结果表明LMP1确实可以诱导cyclinD1的表达 (2～ 4倍 ) ,且诱导具有时间依赖性及剂量依赖性 ;利用三种LMP1功能区缺失的突变体及野生型LMP1,以载体型细胞为对照 ,结合报道基因分析法 ,确定与空白载体细胞系比较 ,野生型LMP1从转录水平可反式激活cyclinD1报道基因活性约 11 2倍 ,其中CTAR1及CTAR2均可活化cyclinD1表达 ,但以CTAR2为主 ,与野生型LMP1诱导cyclinD1反式激活活性比较 ,CTAR1缺失导致cyclinD1报道基因活性下降 2 3 6 % ,CTAR2缺失导致cyclinD1活性下降约 80 7% ,C端均缺失时cyclinD1活性只?
In order to investigate the molecular mechanism of Epstein-Barr virus encoded latent membrane protein 1 (EBV LMP1) promoting cell proliferation and participating in tumorigenesis of EBV-related diseases, it was studied that LMP1 regulates cyclinD1 expression in nasopharyngeal carcinoma cells, thereby affecting cell cycle progression and cell malignancy , And initially identified the domain of LMP1 to play this function.Using Tet on LMP1 HNE2 nasopharyngeal carcinoma cell line has been established, Western blot analysis of LMP1-induced expression of cyclinD1 protein expression kinetics, including time effects and dose effects Three LMP1-deficient mutants and wild-type LMP1 were used to control the expression of LMP1-activated cyclinD1 domain by using vector-type cells as control, and the gene expression and antisense oligodeoxynucleotides were used to block the gene expression The results showed that LMP1 could induce the expression of cyclinD1 (2-4 fold), and the induction of LMP1 in a time-and dose-dependent manner. Using three LMP1 deletion mutants and wild-type LMP1, vector-based cells as a control, combined with the reporter Due to the analysis, wild-type LMP1 transactivates the cyclinD1 reporter gene activity about 11 2 times from that of the blank vector cell line, in which both CTAR1 and CTAR2 can activate cyclinD1 expression, but mainly CTAR2 and wild-type Compared with the LMP1-induced cyclinD1 transactivation activity, the loss of CTAR1 led to a decrease of cyclinD1 reporter gene activity of 236%, CTAR2 deletion resulted in a decrease of cyclinD1 activity of about 80 7%, C-terminal deletion of cyclinD1 activity only?