白色念珠菌生物膜与Toll样受体通路的免疫关系

Immune relationship between candida albicans biofilms and toll-like receptor pathways

目的分析产膜白色念珠菌与感染患者机体Toll样受体(Toll-like receptors,TLRs)通路的免疫关系。方法收集2017年1月至2018年12月间从牡丹江林业中心医院住院血流感染患者标本中分离的白色念珠菌,细胞培养法体外构建生物膜模型,采用结晶紫和荧光染色法定性观察生物膜的生长过程及形态,根据是否产生生物膜将其分为产膜组和非产膜组;采集健康成年人血液样本30份,作为健康对照组。反转录PCR(RT-PCR)检测外周血单个核细胞中TLR2mRNA、TLR4mRNA、髓样分化因子88基因(Myd88mRNA)的表达量变化;免疫印迹法(Western Blot)检测外周血单个核细胞中Myd88蛋白的含量;酶联免疫吸附(ELISA)法测定细胞因子IL-1β、TNF、IFN-γ和IL-4的浓度。结果产膜菌呈菌丝状相互交错密集排列,而非产膜菌大部分以酵母样黏附于培养板底部。产膜组外周血单个核细胞中TLR2mRNA、TLR4mRNA、Myd88mRNA基因的表达量较健康对照组低,非产膜组较健康对照组高(P<0.05)。产膜组外周血单个核细胞中Myd88的含量较健康对照组低,非产膜组较健康对照组高(P<0.05)。产膜组血浆中IL-1β、TNF、IFN-γ浓度与健康对照组相比明显降低,非产膜组与健康对照组相比明显升高(P<0.05);IL-4因子浓度产膜组与健康对照组相比明显升高,非产膜组与健康对照组相比差异无统计学意义(P>0.05)。结论(1)产膜白色念珠菌可能通过下调TLR2mRNA、TLR4mRNA、Myd88mRNA蛋白的表达,使TLR/Myd88/NF-κB信号通路传递受限,增加白色念珠菌的易感性;(2)产膜白色念珠菌可使宿主Th1型免疫反应减弱,导致Th1/Th2比例失衡。以上两点可能是产膜菌相对非产膜菌更容易发生免疫逃逸,并引起机体出现慢性感染的主要原因。

Objective To analyze the immune relationship between membrane-producing candida albicans and toll-like receptor(TLRs)pathways in infected patients.Methods Candida albicans isolated from blood flow infection patients in Mudanjiang Forestry Central Hospital from January 2017 to December 2018 were collected,and a biofilm model was constructed in vitro by cell culture plate method.The growth process and morphology of biofilms were observed by crystal violet and fluorescence staining.It is divided into biofilm group and non-biofilm group according to whether the membrane was produced or not.Thirty blood samples from healthy adults were collected as healthy controls.Changes in the expression of TLR2 mRNA,TLR4 mRNA and myelogenic differentiation factor 88(Myd88mRNA)genes in peripheral blood mononuclear cells of patients with bloodstream infection were detected by reverse transcription PCR(RT-PCR).Western Blot was used to detect the Myd88 protein in peripheral blood mononuclear cells of each group.The concentrations of cytokines il-1,TNF,IFN and il-4 were measured by enzyme-linked immunosorbent assay(ELISA).Results The expression levels of TLR2 mRNA,TLR4 mRNA and Myd88 mRNA in the biofilm-producing group were lower than those in the healthy control group,while those in the non-biofilm group were higher than those in the healthy control group(P<0.05).The content of MyD88 in the biofilm group was lower than that in the healthy control group,while the content of MyD88 in the non-biofilm group was higher than that in the healthy control group(P<0.05).Compared with the healthy control group,the plasma il-1,TNF,IFN concentrations of patients in the biofilm group were significantly reduced,while those in the non-biofilm group were significantly increased(P<0.05).The concentration of il-4 factor in the biofilm group was significantly higher than that in the healthy control group,while the difference between the non-biofilm group and the healthy control group was not statistically significant(P>0.05).Conclusion Membrane producing Candida albicans may inhibit the TLR/Myd88/NF-B signaling pathway and increase candida albicans susceptibility by down-regulating the expression of TLR2 mRNA,TLR4 mRNA and Myd88 mRNA proteins.2.Membrane-producing bacteria can weaken Th1 immune response and induce the body to mainly produce Th2 immune response,leading to the imbalance of Th1/Th2 ratio.The above two points may be the main reasons why the membrane-producing bacteria are more likely to have immune escape than the non-membrane-producing bacteria,and lead to chronic infection in the body.