Dectin-1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：2019-02-26 12:54
【摘要】：目的研究Dectin-1在烟曲霉菌孢子抗原刺激人角膜上皮细胞形成的真菌性角膜炎模型中的固有免疫反应中的作用。 方法用自制的烟曲霉菌孢子抗原刺激培养的人角膜上皮细胞株,以RT-PCR检测各组细胞中Dectin-1 mRNA的表达；用免疫细胞化学方法及酶联免疫吸附试验(ELISA)方法检测细胞中Dectin-1蛋白的表达；以ELISA法检测细胞培养上清中NF-K B、TNF-a、IL-1β、IL-10和CXCL1的浓度。统计学处理采用SPSS 17.0软件进行单因素方差分析。 结果在正常和烟曲霉菌抗原刺激后的人角膜上皮细胞中用RT-PCR均能检测到Dectin-1 mRNA的表达,Dectin-1 mRNA表达随刺激时间增长而逐渐增多,且在2h时达高峰,刺激后各组同空白组相比差距有统计学意义(P=0.000)；用免疫细胞化学方法检测各组细胞中dectin-1蛋白均有表达；ELISA测得HCECs,总蛋白中Dectin-1正常组有表达,刺激后0.5h蛋白含量下降,1h起升高,2h含量达高峰,随后下降,刺激后各组同空白组相比差距有统计学意义(P=0.000)；ELISA法检测各组细胞培养上清中NF-K B刺激后含量增高,2h含量达高峰,刺激后各组同空白组相比差距有统计学意义(P=0.000)；TNF-α,CXCL1和IL-1β刺激后含量增高,分别在刺激2h、4h、1h后达高峰,IL-10刺激后含量增高缓慢,6h增高达高峰,这四个指标刺激后各组同空白组相比差距均有统计学意义(P=0.000)。 结论1、正常的人角膜上皮细胞中存在Dectin-1基因和蛋白水平的表达。 2、用自制的烟曲霉菌孢子抗原刺激正常的人角膜上皮细胞可引起Dectin-1合成量的变化,提示Dectin-1在该真菌性角膜炎模型中可被激活。 3、NF-KB在该真菌性角膜炎模型中表达增加,同时相关炎症因子TNF-a、IL-1β、IL-10和CXCL1的含量变化提示他们也参与了该真菌性角膜炎模型中的免疫反应。
[Abstract]:Aim to study the role of Dectin-1 in the innate immune response of fungal keratitis model induced by spore antigen of Aspergillus fumigatus in human corneal epithelial cells. Methods the cultured human corneal epithelial cells were stimulated with a self-made spore antigen of Aspergillus fumigatus. The expression of Dectin-1 mRNA in the cells of each group was detected by RT-PCR. The expression of Dectin-1 protein was detected by immunocytochemistry and enzyme-linked immunosorbent assay (ELISA), and the concentrations of NF-K-B, TNF-伪, IL-1 尾, IL-10 and CXCL1 in the supernatant of cell culture were detected by ELISA method. One-way ANOVA was analyzed by SPSS 17.0 software. Results the expression of Dectin-1 mRNA was detected by RT-PCR in both normal and Aspergillus fumigatus antigen stimulated human corneal epithelial cells. The expression of Dectin-1 mRNA increased gradually with the increase of stimulation time and reached its peak at 2 h. After stimulation, there was a significant difference between each group and the blank group (P < 0. 000). The expression of dectin-1 protein was detected by immunocytochemistry. The expression of total protein of HCECs, in the normal Dectin-1 group was detected by ELISA. The protein content decreased at 0.5h after stimulation, increased at 1h, reached the peak at 2h, and then decreased. There was a significant difference between each group after stimulation and the blank group (P < 0. 000). ELISA assay was used to detect the content of NF-K B in the supernatant of each group after stimulation, and the content reached the peak at 2 h. There was a significant difference between each group after stimulation and that in the blank group (P < 0. 000). The contents of TNF- 伪, CXCL1 and IL-1 尾 increased after stimulation for 2 h, 4 h and 1 h, respectively. After IL-10 stimulation, the content increased slowly and reached the peak at 6 h. After stimulation, there was a significant difference between each group and the blank group (P < 0. 000). Conclusion 1. The expression of Dectin-1 gene and protein exists in normal human corneal epithelial cells. 2. Stimulation of normal human corneal epithelial cells with a self-made spore antigen of Aspergillus fumigatus induced the change of Dectin-1 synthesis, suggesting that Dectin-1 could be activated in this fungal keratitis model. 3. The expression of NF-B increased in this fungal keratitis model, and the changes of the contents of related inflammatory factors TNF-a,IL-1 尾, IL-10 and CXCL1 suggested that they also participated in the immune response of the fungal keratitis model.
【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
本文编号：2430790
[Abstract]:Aim to study the role of Dectin-1 in the innate immune response of fungal keratitis model induced by spore antigen of Aspergillus fumigatus in human corneal epithelial cells. Methods the cultured human corneal epithelial cells were stimulated with a self-made spore antigen of Aspergillus fumigatus. The expression of Dectin-1 mRNA in the cells of each group was detected by RT-PCR. The expression of Dectin-1 protein was detected by immunocytochemistry and enzyme-linked immunosorbent assay (ELISA), and the concentrations of NF-K-B, TNF-伪, IL-1 尾, IL-10 and CXCL1 in the supernatant of cell culture were detected by ELISA method. One-way ANOVA was analyzed by SPSS 17.0 software. Results the expression of Dectin-1 mRNA was detected by RT-PCR in both normal and Aspergillus fumigatus antigen stimulated human corneal epithelial cells. The expression of Dectin-1 mRNA increased gradually with the increase of stimulation time and reached its peak at 2 h. After stimulation, there was a significant difference between each group and the blank group (P < 0. 000). The expression of dectin-1 protein was detected by immunocytochemistry. The expression of total protein of HCECs, in the normal Dectin-1 group was detected by ELISA. The protein content decreased at 0.5h after stimulation, increased at 1h, reached the peak at 2h, and then decreased. There was a significant difference between each group after stimulation and the blank group (P < 0. 000). ELISA assay was used to detect the content of NF-K B in the supernatant of each group after stimulation, and the content reached the peak at 2 h. There was a significant difference between each group after stimulation and that in the blank group (P < 0. 000). The contents of TNF- 伪, CXCL1 and IL-1 尾 increased after stimulation for 2 h, 4 h and 1 h, respectively. After IL-10 stimulation, the content increased slowly and reached the peak at 6 h. After stimulation, there was a significant difference between each group and the blank group (P < 0. 000). Conclusion 1. The expression of Dectin-1 gene and protein exists in normal human corneal epithelial cells. 2. Stimulation of normal human corneal epithelial cells with a self-made spore antigen of Aspergillus fumigatus induced the change of Dectin-1 synthesis, suggesting that Dectin-1 could be activated in this fungal keratitis model. 3. The expression of NF-B increased in this fungal keratitis model, and the changes of the contents of related inflammatory factors TNF-a,IL-1 尾, IL-10 and CXCL1 suggested that they also participated in the immune response of the fungal keratitis model.
【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
【参考文献】
相关期刊论文 前5条
1 赵文杰;席丽艳;马黎;张军民;李希清;鲁长明;李飞;曾翰翔;;马尔尼菲青霉对巨噬细胞模式识别受体TLR-2、TLR-4、Dectin-1的表达及TNF-α分泌的影响[J];南方医科大学学报;2008年01期
2 荣令;周新;;C型凝集素受体在抗真菌感染中作用的研究进展[J];中国感染与化疗杂志;2008年06期
3 杨策,王正国,蒋建新;模式识别受体Dectin-1与天然免疫反应[J];生理科学进展;2005年02期
4 杨小舟;曾富华;饶力群;;模式识别受体Dectin-1与β-葡聚糖的免疫识别作用[J];生命的化学;2008年05期
5 徐小勇;施毅;;烟曲霉侵袭机制的研究[J];中国呼吸与危重监护杂志;2010年01期
相关博士学位论文 前1条
1 钟文贤;小鼠真菌性角膜炎模型中主要炎性因子表达的研究[D];浙江大学;2007年
相关硕士学位论文 前2条
1 王丽;TLR4信号通路促进肿瘤细胞分泌MIP-3alpha参与肿瘤免疫逃逸的研究[D];浙江大学;2007年
2 李娜;IL-10在大鼠真菌性角膜炎中的表达[D];青岛大学;2010年
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