Dectin-1与TLR2在真菌感染人角膜上皮细胞固有免疫阶段的关系
发布时间：2019-03-06 10:31
【摘要】：目的：研究人永生化角膜上皮细胞(THCE)感染烟曲霉菌菌丝抗原后,THCE细胞表面Dectin-1和TLR2的表达情况,两者在固有免疫阶段的关系,及其对趋化因子的影响。 方法：常规体外培养的THCE细胞为空白对照组,实验组又分为真菌刺激组和通路受体阻断组,用烟曲霉菌菌丝抗原感染体外培养的THCE细胞,建立真菌感染的THCE细胞模型,此为真菌刺激组,通路受体阻断组分别用Dectin-1阻断剂(laminarin)和TLR2中和抗体预先处理THCE细胞30分钟,后加入烟曲霉菌菌丝抗原刺激液,此为Dectin-1阻断+真菌刺激组和TLR2阻断+真菌刺激组,应用实时荧光定量PCR(Real time-PCR)检测4h、8h、16h THCE细胞Dectin-1、TLR2、CXCL1、 CXCL8mRNA的表达；于8h、16h、24h行Western blot检测THCE细胞Dectin-1、 TLR2蛋白的表达。 结果：Dectin-1、TLR2mRNA在真菌菌丝抗原刺激THCE细胞后4h表达升高,8h达高峰,真菌菌丝抗原刺激后8h组与4h、16h组间比较,差异有统计学意义(P0.05)；在真菌菌丝抗原刺激THCE细胞后8h, Dectin-1、TLR2蛋白表达均升高,16h表达量达峰值。趋化因子(CXCL1、CXCL8)的升高趋势与Dectin-1和TLR2的表达趋势一致。laminarin阻断Dectin-1后,真菌菌丝抗原刺激THCE细胞8h, TLR2mRNA表达明显下降(P0.05),而TLR2蛋白在Dectin-1被阻断后16h后,表达也相应下降(P0.05); TLR2中和抗体阻断TLR2后真菌菌丝抗原刺激THCE细胞8h, Dectin-1mRNA表达明显下降(P0.05),其蛋白表达量在阻断TLR2后真菌菌丝抗原刺激16h减少(P0.05)。阻断Dectin-1与阻断TLR2受体,趋化因子(CXCL1、 CXCL8)的表达量均受到明显抑制(P0.05)。 结论：TLR2和Dectin-1共同参与了THCE细胞对烟曲霉菌菌丝抗原体的识别,两者在人角膜上皮细胞抗真菌感染固有免疫阶段存在相互作用,均引起下游趋化因子(CXCL1, CXCL8)的产生,介导角膜上皮细胞的炎症反应。
[Abstract]:Aim: to study the expression of Dectin-1 and TLR2 on the surface of human immortalized corneal epithelial cells (THCE) infected with Aspergillus fumigatus mycelium antigen, the relationship between them in the innate immune stage and their effects on chemokine. Methods: THCE cells were cultured in vitro as blank control group. The experimental group was divided into two groups: fungal stimulation group and pathway blocking group. The cultured THCE cells were infected with Aspergillus fumigatus mycelium antigen to establish the THCE cell model of fungal infection. THCE cells were pretreated with Dectin-1 blocker (laminarin) and TLR2 neutralizing antibody for 30 minutes, and then added Aspergillus fumigatus mycelium antigen stimulation solution. The expression of Dectin-1,TLR2,CXCL1, CXCL8mRNA in THCE cells was detected by real-time fluorescence quantitative PCR (Real time-PCR) at 4 h, 8 h and 16 h in Dectin-1-blocking fungal stimulation group and TLR2-blocking fungal stimulation group. The expression of Dectin-1, TLR2 protein in THCE cells was detected by Western blot at 8 h, 16 h and 24 h. Results: the expression of Dectin-1,TLR2mRNA increased at 4 h and reached a peak at 8 h after stimulation of fungal hyphal antigen in THCE cells. There was significant difference between 8 h group and 4 h group and 16 h group after stimulation of fungal hyphal antigen (P0.05). The expression of Dectin-1,TLR2 protein increased at 8 h after stimulation with fungal hyphae antigen in THCE cells, and reached its peak at 16 h. The increasing trend of chemokine (CXCL1,CXCL8) was consistent with the expression trend of Dectin-1 and TLR2. After Dectin-1 was blocked by laminarin, the expression of TLR2mRNA in THCE cells stimulated by fungal hyphal antigen decreased significantly (P0.05), while the expression of TLR2 protein was decreased 16 hours after Dectin-1 was blocked. The expression also decreased (P0.05). After TLR2 was blocked by TLR2 neutralizing antibody, the expression of Dectin-1mRNA in THCE cells stimulated by fungal hyphal antigen was significantly decreased (P0.05), and the protein expression was decreased at 16h after blocking TLR2 (P0.05). The expression of chemokine (CXCL1, CXCL8) was significantly inhibited by blocking Dectin-1 and TLR2 receptor (P0.05). Conclusion: both TLR2 and Dectin-1 are involved in the recognition of Aspergillus fumigatus mycelium by THCE cells. Both of them interact with each other in the innate immune stage of anti-fungal infection of human corneal epithelial cells, and both lead to the production of downstream chemokine (CXCL1, CXCL8). Mediating inflammation of corneal epithelial cells.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R772.21
本文编号：2435451
[Abstract]:Aim: to study the expression of Dectin-1 and TLR2 on the surface of human immortalized corneal epithelial cells (THCE) infected with Aspergillus fumigatus mycelium antigen, the relationship between them in the innate immune stage and their effects on chemokine. Methods: THCE cells were cultured in vitro as blank control group. The experimental group was divided into two groups: fungal stimulation group and pathway blocking group. The cultured THCE cells were infected with Aspergillus fumigatus mycelium antigen to establish the THCE cell model of fungal infection. THCE cells were pretreated with Dectin-1 blocker (laminarin) and TLR2 neutralizing antibody for 30 minutes, and then added Aspergillus fumigatus mycelium antigen stimulation solution. The expression of Dectin-1,TLR2,CXCL1, CXCL8mRNA in THCE cells was detected by real-time fluorescence quantitative PCR (Real time-PCR) at 4 h, 8 h and 16 h in Dectin-1-blocking fungal stimulation group and TLR2-blocking fungal stimulation group. The expression of Dectin-1, TLR2 protein in THCE cells was detected by Western blot at 8 h, 16 h and 24 h. Results: the expression of Dectin-1,TLR2mRNA increased at 4 h and reached a peak at 8 h after stimulation of fungal hyphal antigen in THCE cells. There was significant difference between 8 h group and 4 h group and 16 h group after stimulation of fungal hyphal antigen (P0.05). The expression of Dectin-1,TLR2 protein increased at 8 h after stimulation with fungal hyphae antigen in THCE cells, and reached its peak at 16 h. The increasing trend of chemokine (CXCL1,CXCL8) was consistent with the expression trend of Dectin-1 and TLR2. After Dectin-1 was blocked by laminarin, the expression of TLR2mRNA in THCE cells stimulated by fungal hyphal antigen decreased significantly (P0.05), while the expression of TLR2 protein was decreased 16 hours after Dectin-1 was blocked. The expression also decreased (P0.05). After TLR2 was blocked by TLR2 neutralizing antibody, the expression of Dectin-1mRNA in THCE cells stimulated by fungal hyphal antigen was significantly decreased (P0.05), and the protein expression was decreased at 16h after blocking TLR2 (P0.05). The expression of chemokine (CXCL1, CXCL8) was significantly inhibited by blocking Dectin-1 and TLR2 receptor (P0.05). Conclusion: both TLR2 and Dectin-1 are involved in the recognition of Aspergillus fumigatus mycelium by THCE cells. Both of them interact with each other in the innate immune stage of anti-fungal infection of human corneal epithelial cells, and both lead to the production of downstream chemokine (CXCL1, CXCL8). Mediating inflammation of corneal epithelial cells.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R772.21
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