白念珠菌ALS3、SSA1基因表达在念珠菌性阴道炎免疫机制中的作用

目的探讨白念珠菌ALS3、SSA1基因缺失对阴道上皮细胞激发免疫反应的作用。方法培养白念珠菌野生株及ALS3、SSA1基因敲除株(SC5314、Δals3、Δssa1),对其进行形态测定。按不同MOI感染人阴道上皮细胞系VK2/E6E7细胞,通过台盼蓝染色观察和乳酸脱氢酶(LDH)活性检测,评价不同MOI白念珠菌对上皮细胞的损伤作用;使用酶联免疫吸附试验(ELISA)评估感染过程中炎性细胞因子及趋化因子在共培养上清中的差异。结果 ALS3基因的缺失对白念珠菌芽管长度影响差异无统计学意义,而SSA1基因的缺失与其他两个菌株相比芽管长度减少约30%～40%(P<0.001)。台盼蓝染色观察及LDH测定发现,3株菌在感染上皮细胞时,其细胞损伤能力均与菌载量成正比;与野生型相比,Δssa1突变体在相同比率感染上皮细胞时,细胞损伤能力明显降低,且差异有统计学意义(P<0.05),Δals3突变株影响较小,甚至略微升高。检测炎性细胞因子及趋化因子发现,突变株在诱导上皮细胞产生促炎因子及趋化因子(GM-CSF、G-CSF、IL-1α、IL-8)的能力上明显减弱,差异均有统计学意义(P<0.05)。结论 ALS3和SSA1基因表达在阴道上皮细胞抗白念珠菌感染的局部免疫应答过程中可能起到重要作用,且SSA1基因表达意义更大。

The role of ALS3 and SSA1 gene expression of Candida albicans in the immune mechanism of vaginal candidiasis

Objective To investigate the effect of ALS3 and SSA1 genes in Candida albicans on immune response of vaginal epithelial cells. Methods Wild strains and ALS3,SSA1 gene knockout strains (SC5314,Δals3,Δssa1) of Candida albicans were cultured and their morphology was determined.Human vaginal epithelial cell line VK2/E6E7 was infected in a certain proportion.The damage of different strains to epithelial cells was evaluated by trypan blue staining and lactate dehydrogenase (LDH) activity detection.The co-culture of inflammatory cytokines and chemokines during infection was evaluated by enzyme-linked immunosorbent assay (ELISA). Results There was no significant difference in the effect of Δals3 on the germ tube length of Candida albicans,while the Δssa1 reduced the germ tube length by about 30%~40% compared with the other two strains (P<0.001).Trypan blue staining and LDH assay showed that the cell damage ability of the three strains was proportional to the fungal load when infected with epithelial cells.Compared with wild strain,the cell damage ability of Δssa1 was significantly reduced when infected with epithelial cells at the same MOI,and the difference was statistically significant (P<0.05),while the impact of Δals3 mutant was weak or even slightly higher.Detection of inflammatory cytokines and chemokines showed that the ability of mutants to induce epithelial cells to produce pro-inflammatory factors and chemokines (GM-CSF,G-CSF,IL-1a,IL-8) was significantly weakened (P<0.05). Conclusion ALS3 and SSA1 gene expression played an important role in the local immune response of vaginal epithelial cells to Candida albicans infection,and SSA1 gene expression was more significant.