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目的观察生殖支原体脂质相关膜蛋白(LAMP)能否诱导宫颈上皮细胞表达人β防御素2(hBD-2),并探讨其可能的调控机制。方法体外培养End1/E6E7人宫颈上皮细胞,用不同浓度的LAMP作用细胞48 h,反转录PCR检测hBD-2 mRNA的表达,Western blot法检测hBD-2蛋白的表达;用Toll样受体2(TLR2)和TLR6中和抗体孵育End1/E6E7细胞,并用TLR2、TLR6和MyD88负显性突变体转染细胞,以明确TLR2、TLR6和MyD88在介导hBD-2表达中的作用;Western blot法检测核因子κB(NF-κB)p65亚基核转位情况,电泳迁移率实验(EMSA)分析LAMP处理前后NF-κB的DNA结合活性;采用NF-κB抑制剂二硫代氨基甲酸吡咯烷(PDTC)处理,观察对hBD-2表达的影响。结果生殖支原体LAMP可诱导End1/E6E7细胞表达hBD-2mRNA和蛋白。TLR2和TLR6中和抗体以及其负显性突变体转染后,能降低hBD-2表达;MyD88负显性突变体也能显著抑制LAMP诱导的hBD-2表达。Western blot结果显示,LAMP处理后可诱导p65核转位,并能增强NF-κB的DNA结合活性。而NF-κB抑制剂PDTC处理后,hBD-2表达降低。结论生殖支原体LAMP可诱导End1/E6E7细胞表达hBD-2,可能受TLR2、TLR6/MyD88/NF-κB调控。
Objective To observe whether LAMP can induce the expression of human β-defensin 2 (hBD-2) in cervical epithelial cells and to explore its possible regulatory mechanism. Methods End1 / E6E7 human cervical epithelial cells were cultured in vitro. The cells were treated with different concentrations of LAMP for 48 h. The expression of hBD-2 mRNA was detected by RT-PCR. The expression of hBD-2 protein was detected by Western blot. Toll- (TLR2) and TLR6 neutralizing antibody to incubate End1 / E6E7 cells, and transfected cells with TLR2, TLR6 and MyD88 negative dominant mutants to clarify the role of TLR2, TLR6 and MyD88 in mediating hBD-2 expression; Western blot The nuclear translocation of NF-κB p65 subunit was detected. The DNA binding activity of NF-κB before and after LAMP treatment was analyzed by electrophoretic mobility shift assay (EMSA). The NF-κB inhibitor dithiocarbamate pyrrolidine PDTC) to observe the impact of hBD-2 expression. Results Mycoplasma genitalium LAMP can induce End1 / E6E7 cells to express hBD-2 mRNA and protein. The transfection of TLR2 and TLR6 neutralizing antibodies and their negative dominant mutants decreased hBD-2 expression. MyD88 negative dominant mutants also significantly inhibited LAMP-induced hBD-2 expression. Western blot results showed that LAMP treatment induced p65 nuclear translocation and enhanced the DNA binding activity of NF-κB. However, the expression of hBD-2 was decreased after NF-κB inhibitor PDTC treatment. Conclusion Mycoplasma genitalium LAMP can induce End1 / E6E7 cells to express hBD-2, which may be regulated by TLR2, TLR6 / MyD88 / NF-κB.