Dectin-1介導(dǎo)的人角膜上皮細(xì)胞對煙曲霉菌的固有免疫反應(yīng)
發(fā)布時間：2019-02-26 12:54
【摘要】：目的研究Dectin-1在煙曲霉菌孢子抗原刺激人角膜上皮細(xì)胞形成的真菌性角膜炎模型中的固有免疫反應(yīng)中的作用。 方法用自制的煙曲霉菌孢子抗原刺激培養(yǎng)的人角膜上皮細(xì)胞株,以RT-PCR檢測各組細(xì)胞中Dectin-1 mRNA的表達；用免疫細(xì)胞化學(xué)方法及酶聯(lián)免疫吸附試驗(ELISA)方法檢測細(xì)胞中Dectin-1蛋白的表達；以ELISA法檢測細(xì)胞培養(yǎng)上清中NF-K B、TNF-a、IL-1β、IL-10和CXCL1的濃度。統(tǒng)計學(xué)處理采用SPSS 17.0軟件進行單因素方差分析。 結(jié)果在正常和煙曲霉菌抗原刺激后的人角膜上皮細(xì)胞中用RT-PCR均能檢測到Dectin-1 mRNA的表達,Dectin-1 mRNA表達隨刺激時間增長而逐漸增多,且在2h時達高峰,刺激后各組同空白組相比差距有統(tǒng)計學(xué)意義(P=0.000)；用免疫細(xì)胞化學(xué)方法檢測各組細(xì)胞中dectin-1蛋白均有表達；ELISA測得HCECs,總蛋白中Dectin-1正常組有表達,刺激后0.5h蛋白含量下降,1h起升高,2h含量達高峰,隨后下降,刺激后各組同空白組相比差距有統(tǒng)計學(xué)意義(P=0.000)；ELISA法檢測各組細(xì)胞培養(yǎng)上清中NF-K B刺激后含量增高,2h含量達高峰,刺激后各組同空白組相比差距有統(tǒng)計學(xué)意義(P=0.000)；TNF-α,CXCL1和IL-1β刺激后含量增高,分別在刺激2h、4h、1h后達高峰,IL-10刺激后含量增高緩慢,6h增高達高峰,這四個指標(biāo)刺激后各組同空白組相比差距均有統(tǒng)計學(xué)意義(P=0.000)。 結(jié)論1、正常的人角膜上皮細(xì)胞中存在Dectin-1基因和蛋白水平的表達。 2、用自制的煙曲霉菌孢子抗原刺激正常的人角膜上皮細(xì)胞可引起Dectin-1合成量的變化,提示Dectin-1在該真菌性角膜炎模型中可被激活。 3、NF-KB在該真菌性角膜炎模型中表達增加,同時相關(guān)炎癥因子TNF-a、IL-1β、IL-10和CXCL1的含量變化提示他們也參與了該真菌性角膜炎模型中的免疫反應(yīng)。
[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-偽, 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- 偽, 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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R772.21
本文編號：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-偽, 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- 偽, 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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R772.21
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