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构建表达DR4的质粒pCMV-DR4-HA(DR4)和表达PRMT5的质粒pCMV-PRMT5-Flag,共转染293T细胞,验证DR4和PRMT5的相互作用.将DR4和蛋白精氨酸N端甲基化转移酶5(protein arginine methyltransferase5,PRMT5)两种质粒分别或者共转染293T细胞,研究PRMT5对DR4引起的炎症因子释放的影响,探讨PRMT5抑制DR4(tumor necrosis factor-related apoptosis-inducing ligand receptor1)引起炎症因子释放的分子机制.分别通过RT-PCR和ELISA方法对炎症因子的表达进行定量检测.通过双荧光报告基因方法检测PRMT5对DR4引起NF-κB活性变化的影响,并且使用WesternBlot方法检测ERK的表达变化.DR4和PRMT5在293T细胞中能相互结合,PRMT5过表达降低了DR4引起的NF-κB活性和ERK的磷酸化,导致CCL20分泌减少.因此,PRMT5在293T细胞中与DR4结合,通过改变NF-κB和ERK激酶活性影响了CCL20的分泌,参与了DR4介导的细胞免疫调节.
Construction of DR4-expressing plasmid pCMV-DR4-HA (DR4) and PRMT5-expressing plasmid pCMV-PRMT5-Flag were co-transfected into 293T cells to verify the interaction of DR4 and PRMT5 N-terminal methylation of DR4 and protein arginine To investigate the effect of PRMT5 on the release of inflammatory cytokines induced by DR4 in 293T cells, and to investigate the effect of PRMT5 on tumor necrosis factor-related apoptosis-inducing ligand receptor1 The molecular mechanism of the release of inflammatory cytokines was analyzed by RT-PCR and ELISA respectively.The effect of PRMT5 on NF-κB activity induced by DR4 was detected by double fluorescent reporter gene assay, and Western blotting was used to detect the expression of ERK , And PRMT5 overexpression decreased the NF-κB activity and ERK phosphorylation induced by DR4, leading to the decrease of CCL20 secretion.Therefore, PRMT5 binds to DR4 in 293T cells by changing NF-|ÊB and ERK kinase activities affect the secretion of CCL20 and are involved in DR4-mediated cellular immune regulation.