人角质形成细胞Cx26-G12R突变可下调TLR2及下游炎症因子的表达

G12R mutation in Cx26 downregulates the expression levels of TLR2 and its downstream inflammatory factors in keratinocytes

目的探讨人角质形成细胞Cx26发生G12R突变对TLR2及下游炎症因子表达的影响。方法将野生型Cx26(WT-Cx26)和G12R突变(G12R-Cx26)的人角质形成细胞(HaCaT)分别与红色毛癣菌孢子共培养。采用实时荧光定量PCR法检测TLR2及其下游效应分子的mRNA表达水平;Western blot法检测TLR2表达情况;ELISA法检测细胞因子的分泌;细胞免疫荧光检测Cx26和TLR2内化情况。结果与WT-Cx26细胞株相比,G12R-Cx26突变株中,TLR2的mRNA水平无明显变化(t=1.92,P=0.190),而细胞膜和胞质中的TLR2蛋白水平均下降(均P<0.01);TLR2通路下游炎症因子TNF(t=6.91,P<0.05)、IL-6(t=13.03,P<0.01)和抗菌肽LL-37(t=25.31,P<0.001)的表达下调。与空白对照组相比,添加内化抑制剂的G12R-Cx26 HaCaT细胞Cx26与TLR2在细胞膜上的共定位增加。结论人角质形成细胞中Cx26-G12R突变可下调TLR2及下游炎症因子TNF、IL-6和抗菌肽LL-37的表达。

Objective To investigate the effect of G12R mutation in Cx26 on the expression of TLR2 and its downstream inflammatory factors in human keratinocytes.Methods Human keratinocytes(HaCaT)without(WT-Cx26)or with G12R-mutation(G12R-Cx26)were co-cultured with Trichophyton rubrum.Expression levels of TLR2 mRNA and its downstream effector molecules were detected by real-time PCR.The expression levels of TLR2 protein were detected by western blot.Cytokine levels in the culture medium were measured by ELISA.Immunofluorescence was used to determine distribution of Cx26 and TLR2 in G12R-Cx26 HaCaT cells.Results Expression levels of TLR2 mRNA were comparable between WT-Cx26 and G12R-Cx26 cells(t=1.92,P=0.190).However,G12R mutation in Cx26 decreased expression levels of TLR2 protein in cell membrane and cytoplasm(both P<0.01 vs.WT-Cx26)in addition to reductions in expression levels of downstream of TLR2 pathway,including TNF(t=6.91,P<0.05),IL-6(t=13.03,P<0.01)and antimicrobial peptide LL-37(t=25.31,P<0.001).Coculture of G12R-Cx26 cells with ikarugamycin increased co-localization of Cx26 and TLR2 on the cell membrane.Conclusion Cx26-G12R mutation down-regulates the expression of TLR2 and downstream inflammatory cytokines,including TNF,IL-6 and antimicrobial peptide LL-37,in keratinocytes.