EBV LMP1对牙龈上皮细胞促炎因子的调控及机制研究

Regulation and Mechanism of EBV LMP1 on Inflammatory Factors of Gingival Epithelial Cells

目的:探究EB病毒潜在膜蛋白1(Epstein-barr virus latent membrane protein 1,EBV LMP1)对牙龈上皮细胞促炎因子的调控及机制。方法:0.05μg和0.2μg pSG-GFP-LMP1质粒转染Ca9-22细胞,分别于24 h和48 h后荧光显微镜下观察GFP在Ca9-22细胞中的表达情况,Real-time PCR和Western blot检测LMP1 mRNA和蛋白表达,确定转染是否成功。应用0.2μg pSG-GFP-LMP1质粒转染Ca9-22细胞,分别于24 h和48 h进行Real-time PCR和ELISA检测细胞中IL-8 mRNA和蛋白表达。应用0.05μg和0.2μg pSG-GFP-LMP1质粒转染Ca9-22细胞,48 h后Real-time PCR和ELISA检测细胞中IL-8 mRNA和蛋白表达,Western blot检测p-IκBα、IκBα、p-p65和p65蛋白表达。结果:pSG-GFP-LMP1质粒转染Ca9-22细胞基因高表达,转染成功。0.2μg质粒转染Ca9-22细胞24 h和48 h后,实验组IL-8 mRNA和蛋白表达均显著高于对照组(P<0.01)。0.05μg和0.2μg质粒转染Ca9-22细胞48 h后,实验组IL-8 mRNA和蛋白表达以及p-IκBα、p-p65和p65蛋白表达显著高于对照组(P<0.01),且随着剂量增大,表达增强,实验组IκBα蛋白表达显著低于对照组(P<0.01),且随着剂量增大,表达减弱。结论:EBV LMP1可能通过激活NF-κB磷酸化进而诱导Ca9-22细胞中IL-8的表达。

Objective:To study the regulation of EBV LMP1 on proinflammatory factors of Ca9-22 cells as well as its mechanism.Methods:Ca9-22 cells were transfected with 0.05μg and 0.2μg pSG-GFP-LMP1 plasmids.The expressions of GFP were observed under fluorescence microscope,the mRNA and protein levels of LMP1 were measured by Real-time PCR and ELISA after 24 h and 48 h,respectively.Ca9-22 cells were transfected with 0.2μg pSG-GFP-LMP1 plasmids,and the mRNA and protein levels of IL-8 were measured by real-time PCR and ELISA at 24 h and 48 h.Ca9-22 cells were transfected with 0.05μg and 0.2μg pSG-GFP-LMP1 plasmids,the protein levels of p-IκBα,IκBα,p-p65,and p65 were measured by western blot.Results:Ca9-22 cells transfected with the pSG-GFP-LMP1 plasmid could be expressed successfully.After transfection of Ca9-22 cells with 0.2μg plasmid,the mRNA and protein levels of IL-8 in the experimental group were significantly increased(P<0.01).48h after transfection,the mRNA and protein levels of IL-8,the protein levels of p-IκBα,p-p65,and p65 in the experimental group were significantly increased(P<0.01).Furthermore,the expressions were increased in a dose-dependent manner.The protein levels of IκBαwas decreased in the experimental group(P<0.01).Conclusion:EBV LMP1 may induce IL-8 expression in Ca9-22 cells by activating NF-κB phosphorylation.