MBL介导的人角膜上皮细胞对烟曲霉菌的固有免疫反应
发布时间：2018-01-13 16:05
本文关键词：MBL介导的人角膜上皮细胞对烟曲霉菌的固有免疫反应 出处：《青岛大学》2011年硕士论文 论文类型：学位论文
【摘要】：目的研究甘露糖结合凝集素(mannan-binding lectin, MBL)在体外培养的人角膜上皮细胞(HCECs)中的表达,及用自制烟曲霉菌孢子抗原刺激液刺激后的细胞中MBL及炎症细胞因子的表达变化,分析MBL在角膜烟曲霉菌感染中的作用。 方法体外培养的HCECs细胞增殖达到80%-90%融合时,弃掉培养基,更换为无血清培养基培养24h后,实验组加自制的灭活的烟曲霉菌孢子抗原刺激液,分别于刺激的0h、0.5h、1h、2h、4h、6h、8h收取细胞,逆转录RT-PCR检测正常HCECs及烟曲霉菌孢子抗原刺激液刺激后的HCECs中MBL mRNA的表达；酶联免疫吸附试验(ELISA)方法检测正常HCECs及烟曲霉菌孢子抗原刺激液刺激后的HCECs中MBL的浓度及其HCECs培养上清中MBL、NF-κB、IL-1β、TNF-α、IL-6和IL-10的浓度,观察刺激不同时间后各组HCECs培养上清中MBL、NF-κB、IL-1β、TNF-α、IL-6和IL-10的含量变化。 结果正常HCECs有微量MBL mRNA的表达。烟曲霉菌孢子抗原刺激HCECs后,MBL mRNA表达逐渐增多,在刺激2h时达高峰,之后逐渐下降,与正常HCECs比较差异具有显著性(P0.05)；ELISA测得HCECs中MBL在刺激后2h含量达高峰,与正常HCECs比较差异具有显著性(P0.05)；HCECs培养上清中MBL表达量逐渐下降,刺激后各时间点与刺激0.5h后HCECs比较差异具有显著性(P0.05)；NF-κB、IL-1β、TNF-α、IL-6和IL-10分别在刺激2h、1h、2h、4h、6h后含量达高峰,各时间点与正常HCECs比较差异具有显著性(P0.05)。 结论1.体外培养的人角膜上皮细胞可表达MBL。2.MBL参与了人角膜上皮细胞对烟曲霉菌孢子抗原的识别。3.MBL可介导人角膜上皮细胞NF-κB通路的活化并可诱导下游炎性细胞因子TNF-α、IL-β、IL-6和IL-10的表达,提示MBL可在角膜抵御真菌感染早期固有免疫中起作用。
[Abstract]:Objective to investigate the expression of mannose binding lectin (MBL) in cultured human corneal epithelial cells (HCECs). The expression changes of MBL and inflammatory cytokines in the cells stimulated by self-made Aspergillus fumigatus spore antigen were analyzed to analyze the role of MBL in the infection of Aspergillus fumigatus. Methods when the proliferation of HCECs cells reached 80% -90% in vitro, the culture medium was abandoned and replaced with serum-free medium for 24 hours. In the experimental group, the inactivated aspergillus fumigatus spore antigen was added to the stimulation solution, and the cells were collected at 0. 5 h, 0. 5 h, 1 h, 2 h, 4 h, 4 h, 6 h and 8 h, respectively. The expression of MBL mRNA in normal HCECs and HCECs stimulated by Aspergillus fumigatus spore antigen was detected by reverse transcription RT-PCR. Enzyme linked immunosorbent assay (Elisa) was used to detect the concentration of MBL in normal HCECs and HCECs stimulated by Aspergillus fumigatus spore antigen and MBL in the supernatant of HCECs culture. The concentrations of TNF- 伪 -IL-6 and IL-10 in the supernatant of HCECs cultured in each group were observed after stimulation for different time, and the levels of IL-1 尾 in the supernatant of HCECs were observed. The contents of IL-6 and IL-10 in TNF- 伪 were changed. Results there was a small amount of MBL mRNA expression in normal HCECs, and the expression of mRNA increased gradually after HCECs was stimulated by Aspergillus fumigatus spore antigen, and reached its peak at 2h after stimulation. Then it decreased gradually, and the difference was significant compared with the normal HCECs (P 0.05). The content of MBL in HCECs reached its peak at 2 h after stimulation by ELISA, and the difference was significant compared with that of normal HCECs (P 0.05). The expression of MBL in the supernatant of HCECs decreased gradually, and there was a significant difference in HCECs between the two groups at each time point after stimulation and 0.5 h after stimulation (P 0.05). NF- 魏 B, IL-1 尾, TNF- 伪, IL-6 and IL-10 reached their peak levels at 2h, 1h, 2h, 2h, 4h and 6h, respectively. There was a significant difference in HCECs between each time point and normal group (P 0.05). Conclusion 1.Human corneal epithelial cells can express MBL.2.MBL in vitro and participate in the recognition of Aspergillus fumigatus spore antigen by human corneal epithelial cells. 3. MBL can mediate the expression of MBL.2.MBL in human corneal epithelial cells. Activation of 魏 B pathway can induce down-stream inflammatory cytokine TNF- 伪. The expression of IL-尾-6 and IL-10 suggests that MBL may play a role in the early innate immunity of cornea against fungal infection.
【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】：R392
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