表皮生长因子受体在阴道念珠菌病发病过程中的作用初探

The role of EGFR in the pathogenesis of vaginal candidiasis

目的初步探索表皮生长因子受体(epidermal growth factor receptor,EGFR)在阴道念珠菌病发病过程中的作用及分子机制。方法培养阴道上皮细胞(VK2/E6E7细胞),白念珠菌刺激后利用荧光定量PCR检测EGFR及可能相关的免疫通路因子的表达。构建EGFR-siRNA VK2/E6E7细胞模型,与白念珠菌共培养后,分别利用ELLISA及全自动细胞检测仪检测表皮生长因子受体敲除前后阴道上皮细胞分泌细胞因子的变化以及对白念珠菌感染防御能力的改变。构建阴道念珠菌感染小鼠模型,qPCR检测阴道组织表皮生长因子受体及免疫通路因子的表达;并检测EGFR磷酸化抑制剂阻断通路后,阴道组织局部的真菌载量和炎性细胞的变化。结果qPCR检测显示,EGFR、STAT3、GM-CSF及IL^-1β在VK2/E6E7细胞感染白念珠菌后表达升高,且具有统计学意义(P<0.05);ELLISA检测结果显示,EGFR-siRNA VK2/E6E7细胞感染白念珠菌后GM-CSF、IL-8、IL^-1β、MIP-3α表达显著下降(P<0.05);全自动细胞检测仪检测结果显示,EGFR敲除细胞在感染白念珠菌30 h后的防御能力和细胞活性较正常细胞明显降低。qPCR检测显示小鼠阴道感染白念珠菌后EGFR、HER2、STAT3、IL-8表达升高具有统计学意义(P<0.05);局部应用磷酸化抑制剂阻断EGFR通路后,小鼠阴道灌洗液菌载量较对照组明显增加,且在感染后第7日差异有统计学意义(P<0.05)。结论表皮生长因子受体及其通路因子在阴道念珠菌病发病过程中起到较为重要的作用。

Objective To investigate the role and molecular mechanism of EGFR in the pathogenesis of vaginal candidiasis.Methods Vaginal epithelial cells(VK2/E6E7 cells)were cultured.After stimulation with Candida albicans,the expression of immune factors in EGFR and possible related immune pathway factors were detected by real-time PCR.The EGFR-siRNA VK2/E6E7 cell model was constructed and co-cultured with Candida albicans.The cytokines secreted by vaginal epithelial cells and the protective ability against Candida albicans infection were detected by ELLISA and automatic cell detector.A mouse model of vaginal Candida infection was constructed.qPCR was used to detect the expression of EGFR and immune pathway factors in vaginal tissues.The changes of fungal load and inflammatory cells in vaginal tissues were detected after EGFR phosphorylation inhibitor blocking pathway.Results qPCR assay showed that EGFR,STAT3,GM-CSF and IL^-1βwere up-regulated in VK2/E6E7 cells infected with Candida albicans,and were statistically significant(P<0.05).ELLISA assay showed the expressions of GM-CSF,IL-8,IL^-1βand MIP-3αwere significantly decreased in EGFR-siRNA VK2/E6E7 cells infected with Candida albicans(P<0.05).The results of automatic cell detector showed that the defense capacity and cell activity of EGFR knockout cells after 30 hours’infection of Candida albicans were significantly lower than normal cells.The results of qPCR showed that the expression of EGFR,HER2,STAT3 and IL-8 following the vaginal infection of Candida albicans in the mouse was statistically significant(P<0.05);after local application of phosphorylation inhibitor,the lavage fluid load in the mouse vagina was significantly higher than that of the control group with EGFR pathway being blocked and there was a statistical difference on the 7th day after infection(P<0.05).Conclusion The EGFR and its pathway factors should play an important role in the pathogenesis of vaginal candidiasis.