不同Sap酶活性的白假丝酵母菌对阴道上皮细胞表达NLRP3炎性小体的影响

Effect of Candida albicans with different Sap enzyme activities on NLRP3 inflammasome expression in vaginal epithelial cells

目的研究不同分泌型天冬氨酸蛋白酶(Sap)酶活性的白假丝酵母菌对人和小鼠阴道上皮细胞表达核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性小体的影响。方法收集外阴阴道假丝酵母菌病(VVC)患者阴道分泌物后纯化、鉴定菌株、检测Sap酶活性;酶分步消化法培养人和小鼠阴道上皮细胞并与不同Sap酶活性的白假丝酵母菌共培养;用免疫荧光法检测阴道上皮细胞中NLRP3炎性小体的表达;用ELISA法检测细胞上清液中白细胞介素(IL)-1β、IL-18的水平;用HE染色观察细胞形态。人和小鼠阴道上皮细胞培养方式基本一致。结果Sap酶活性强组NLRP3炎性小体、IL-1β和IL-18水平均高于Sap酶活性弱组,并且在感染早期即达高峰。随着感染时间增加可看到细胞破裂、死亡,NLRP3炎性小体也随之下降,这在小鼠和人的阴道上皮细胞中呈现出相同趋势。结论白假丝酵母菌可以刺激阴道上皮细胞表达更多的NLRP3炎性小体、IL-1β和IL-18,Sap酶活性强的白假丝酵母菌作用更强;小鼠和人阴道上皮细胞感染后的炎症反应一致,因此小鼠可代替人作为VVC体内研究的模式动物。

Objective To study the effect of Candida albicans with different Sap enzyme activities on the expression of NLRP3 inflammasome in humans and mice vaginal epithelial cells.Methods Candida collected from the vagina of vulvovaginal candidiasis(VVC)patients was purified and identified,and the Sap enzyme activity was detected;Humans and mice vaginal epithelial cells were cultured by enzyme stepwise digestion and co-cultured with Candida albicans with different Sap enzyme activities;The expression of NLRP3 inflammasome was detected in vaginal epithelial cells by immunofluorescence method;The levels of interleukin(IL)-1βand IL-18 in cell supernatant was detected by ELISA method;Cell morphology was observed by HE staining.The culture methods of humans and mice vaginal epithelial cells were in the same way.Results The levels of NLRP3 inflammasome,IL-1βand IL-18 in the group with strong Sap activity were all higher than those in the group with weak Sap activity,and reached their peaks in the early stage of infection.As the infection time increases,cells could be seen to rupture and die,and NLRP3 inflammasomes also declined,this presented the same tendency in humans and mice vaginal epithelial cells.Conclusion Candida albicans could stimulate the expression of NLRP3 inflammasome,IL-1βand IL-18 in vaginal epithelial cells,and Candida albicans with strong Sap activity was more effective.Humans and mice vaginal epithelial cells had the same inflammatory response after infection,so mice could replace humans as the model animal for VVC in vivo research.