表面活性蛋白A、D在烟曲霉菌感染人角膜上皮细胞炎症反应中作用的实验研究

发布时间：2018-05-03 14:50

本文选题：角膜上皮 + 烟曲霉菌　；参考：《青岛大学》2011年硕士论文

【摘要】：目的：研究表面活性蛋白A、D(surfactant protein A and D,SP-A、SP-D)在烟曲霉菌(Aspergil lus fumigatus,A. fumigatus,AF)性角膜炎早期固有免疫应答阶段的表达变化和AF感染永生化人角膜上皮细胞(tolerated human corneal epithelial, THCEs)炎症反应中SP-A、SP-D的作用。方法：体外培养THCEs,自制烟曲霉菌孢子抗原刺激液,不同时间点0.5h、1h、2h、4h、6h和8h刺激各组THCEs(n=6)。以逆转录聚合酶链式反应(Reversed-transcript PCR, RT-PCR)检测THCEs中SP-A、SP-D mRNA的表达；以ELISA检测THCEs培养液上清中SP-A、SP-D的蛋白质表达水平、NF-kB的活化情况及下游Thl型细胞因子TNF-a、IL-1β和Th2型细胞因子IL-8、IL-10的变化。免疫细胞化学法检测THCEs中SP-A、SP-D的表达部位和表达强弱情况。结果：正常THCEs中有SP-A、SP-D mRNA和蛋白质的微量表达。烟曲霉菌孢子抗原以时间依赖方式促进THCEs中SP-A、SP-D的表达及细胞因子的分泌。(1)给予烟曲霉菌孢子抗原刺激0.5h后,SP-A、SP-D mRNA表达水平开始升高,分别为正常细胞组(对照组)的1.29倍和1.02倍(P0.01); SP-A、SP-D mRNA表达水平2h达最高峰,分别为对照组的3.00倍和1.57倍(P0.01)。(2)细胞上清SP-A、SP-D蛋白表达量在给予烟曲霉菌孢子抗原刺激0.5h后增多,分别为对照组的1.05倍和0.91倍(P0.01),2h表达量最多,分别为对照组的1.48倍和1.92倍(P0.01)。烟曲霉菌孢子抗原刺激后,NF-kB活化,并与SP-A、SP-D表达量呈负相关。烟曲霉菌孢子抗原刺激THCEs 1h组TNF-a、IL-1β表达开始增多,分别为对照组的1.82倍、1.43倍(P0.05),刺激2h组表达量达最高峰,分别为对照组的2.80倍、2.86倍(P0.01)。IL-8、IL-10在烟曲霉菌孢子抗原刺激1h组表达量开始升高,分别为对照组的1.12倍、1.28倍(P0.05),表达量呈时间依赖方式逐渐升高。(3)免疫细胞化学检测发现,正常THCEs胞浆中有SP-A、SP-D的弱阳性表达,给予烟曲霉菌孢子抗原刺激0.5h开始阳性表达呈时间依赖性逐渐增强。结论：1、在烟曲霉菌感染早期阶段,SP-A和SP-D参与了THCEs对烟曲霉菌孢子抗原的识别。2、SP-A、SP-D在真菌性角膜炎固有免疫早期阶段发挥重要保护性作用,他们与炎性因子、免疫细胞共同构成了角膜抵御真菌的免疫防线。
[Abstract]:Objective: to investigate the expression of surface-active protein A and DX SP-AP-D in the early stage of innate immune response to Aspergil lus fumigatus. AFA) and the role of SP-AP-D in the inflammatory response of human corneal epithelial cells infected with AF to immortalized human corneal epithelial cells (human corneal epithelial, THCEs). Methods: the expression of SP-AP-D in the early stage of chronic keratitis induced by Aspergil lus fumigatusa (Afumigatusa) and the role of SP-AP-D in the inflammatory response of human corneal epithelial cells infected with AF were studied. Methods: THCs were cultured in vitro and stimulated by Aspergillus fumigatus spore antigen at different time points of 0.5 h, 1 h, 2 h, 4 h, 6 h and 8 h, respectively. Reverse transcriptase chain reaction (RT PCR) was used to detect the expression of SP-An SP-D mRNA in THCEs, and ELISA was used to detect the protein expression level of SP-An SP-D in the supernatant of THCEs. The activation of NF-kB and the changes of the downstream Thl cytokines TNF-a IL-1 尾 and Th2 type IL-8 IL-10 were detected. Immunocytochemistry was used to detect the expression of SP-An SP-D in THCEs. Results: SP-An SP-D mRNA and protein were expressed in normal THCEs. Aspergillus fumigatus spore antigen promoted the expression of SP-An SP-D and cytokine secretion in THCEs in a time-dependent manner. The expression level of SP-An SP-D mRNA began to increase after stimulation with Aspergillus fumigatus spore antigen for 0.5 h. The expression level of SP-AP-D mRNA reached its highest level at 2h, which was 3.00 and 1.57 times of that of the control group, respectively. The expression of SP-AnSP-D protein in the supernatant of Aspergillus fumigatus was increased after 0.5 h of stimulation with Aspergillus fumigatus spores antigen, and the expression of SP-AmSP-D protein in the supernatant of the control group was 1.29 times and 1.02 times as much as that of the control group, respectively. It was 1.05 times and 0.91 times as much as that of the control group for 2 hours, and 1.48 times and 1.92 times of the control group, respectively, and the expression of P0.01C was 1.48 times and 1.92 times of that of the control group, respectively. The activation of NF-kB by Aspergillus fumigatus spore antigen was negatively correlated with the expression of SP-An SP-D. The expression of IL-1 尾 in THCEs treated with Aspergillus fumigatus spore antigen for 1 h began to increase, which was 1.82 times as much as that in control group (1.43 times, P 0.05), and reached the highest level in group 2 h. The expression of IL-10 began to increase in the group stimulated by Aspergillus fumigatus spore antigen for 1 h, 1.12 times and 1.28 times of the control group, respectively. The expression of IL-10 increased gradually in a time-dependent manner. The immunocytochemistry showed that the expression of IL-10 was increased gradually in a time-dependent manner. There was a weak positive expression of SP-An SP-D in the cytoplasm of normal THCEs, and the expression of SP-D was gradually increased in a time-dependent manner at 0.5 h after stimulation with Aspergillus fumigatus spores antigen. ConclusionSP-A and SP-D are involved in the early stage of aspergillus fumigatus infection. SP-A and SP-D play an important protective role in the early stage of innate immunity of fungal keratitis by THCEs recognition of Aspergillus fumigatus spore antigen. Immune cells together form the immune defense line against fungi in the cornea.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R772.21

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