白色念珠菌感染诱导人气道上皮细胞的损伤机制  

Mechanism of human airway epithelial cell injury induced by Candida albicans infection

作　　者：史清梅 杨丹丹 孟繁君 杨晓燕 王利新 Shi Qingmei;Yang Dandan;Meng Fanjun;Yang Xiaoyan;Wang Lixin(Department of Laboratory Medicine,Hospital of Cardiovascular and Cerebrovascular Diseases of General Hospital of Ningxia Medical University,Yinchuan 750001,China;College of Life Science,Ningxia University,Yinchuan 750021,China)

机构地区：[1]宁夏医科大学总医院心脑血管病医院医学检验科,银川750001 [2]宁夏大学生命科学学院,银川750021

出　　处：《中华医学杂志》2022年第25期1924-1930,共7页National Medical Journal of China

基　　金：宁夏自然科学基金(2019AAC03221)。

摘　　要：目的探讨不同产膜能力白色念珠菌入侵后诱导人气道上皮细胞的免疫损伤机制。方法选择2019年6至12月宁夏医科大学总医院分离培养的25株白色念珠菌,质控菌株SC5314为标准菌株。建立白色念珠菌生物膜体外模型,利用结晶紫染色和酶标板法检测不同白色念珠菌的生物膜形成能力;采用酶标板法测定570 nm处的吸光度值(A_(570)):A_(570)≥0.5为强产膜菌(SBF),0.25<A_(570)<0.5为中产膜菌(DRF),A_(570)≤0.25为不产膜菌(WBF)。在体外分离培养并建立人气道上皮细胞的气液相培养模型,分为5组:空白对照组(n=20)、标准菌株组(n=20)、强产膜组(n=19)、不产膜组(n=17)、耐氟康唑组(n=18)。扫描电镜观察气液相培养上皮细胞的形态。采用免疫荧光检测气液相培养上皮细胞体外模型标志蛋白的表达。通过微孔板法检测细胞中乳酸脱氢酶(LDH)含量,利用酶联免疫吸附实验(ELISA)检测细胞培养液中细胞内β-防御素(hBD2)、粒巨噬细胞集落刺激因子(GM-CSF)、粒细胞集落刺激因子(G-CSF)等的分泌情况。结果强产膜菌株以菌丝相互交错生长,能看到极少数酵母细胞包裹于其中。扫描电镜观察气液相培养的上皮细胞菌丝能够主动入侵上皮细胞;纤毛乙酰化的微管蛋白和角蛋白的表达量明显减少,同时细胞增殖相关蛋白的表达也被下调。空白对照组、标准菌株组、强产膜组、不产膜组、耐氟康唑组细胞中LDH含量分别为(12.21±5.68)、(46.35±6.35)、(18.69±4.38)、(12.56±3.69)、(13.48±4.28)U/L,差异有统计学意义(P<0.001);与标准菌株组相比,强产膜组、不产膜组、耐氟康唑组细胞内LDH含量均降低(均P<0.01)。空白对照组、标准菌株组、强产膜组、不产膜组、耐氟康唑组细胞内hBD2的含量分别为(26.14±0.77)、(56.18±0.83)、(30.66±2.59)、(29.22±0.48)、(28.28±1.56)ng/L,差异有统计学意义(P<0.001);与空白对照组相比,只有标准菌株组的上皮细�Objective To investigate the immune mechanism of human airway epithelial cell injury induced by invasion of Candida albicans with different biofilm formation abilities.Methods Twenty-five strains of Candida albicans isolated and cultured in General Hospital of Ningxia Medical University from June to December 2019 were selected,and quality control strain SC5314 was used as the standard strain.An in vitro model of Candida albicans biofilm was established,and the biofilm formation ability of different Candida albicans was detected by crystal violet staining and enzyme plate method.The absorbance value at 570 nm(A_(570))was determined by enzyme plate method.A_(570)≥0.5,0.25<A_(570)<0.5 and A_(570)≤0.25 indicated strong biofilm Candida albicans form(SBF),moderate biofilm Candida albicans form(DRF)and weak biofilm Candida albicans form(WBF),respectively.The gas-liquid phase culture model of human airway epithelial cells was isolated and established in vitro and divided into five groups,including blank control group(n=20),standard strain group(n=20),strong biofilm group(n=19),weak biofilm group(n=17)and fluconazole-resistant group(n=18).The morphology of the epithelial cells was observed by scanning electron microscope(SEM),and the expression of marker protein in the model was detected by immunofluorescence in vitro.The level of lactate dehydrogenase(LDH)in cells was detected by microplate method,and the secretion ofβ-defensin(hBD2),granulocyte macrophage colony stimulating factor(GM-CSF)and granulocyte colony-stimulating factor(G-CSF)was detected by enzyme-linked immunosorbent assay(ELISA).Results The strong biofilm strains grew with interlacing mycelia,and very few yeast cells could be seen wrapped in them.SEM observed that the mycelia of epithelial cells in gas-liquid phase culture could actively invade epithelial cells,and the expression of acetylated tubulin and keratin in cilia were significantly reduced,while the expression of Ki67 was down-regulated.The LDH levels in the blank control group,standard strain

关 键 词：念珠菌 白色 人气道上皮细胞 生物膜 损伤机制 

分 类 号：R446.5[医药卫生—诊断学]