烟曲霉菌性角膜炎中LOX-1和TLR4相互作用调节ROS生成的研究

发布时间：2018-04-21 23:03

本文选题：烟曲霉菌 + 角膜炎　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:为了探讨烟曲霉菌性角膜炎中LOX-1和TLR4的关系,证明在烟曲霉菌性角膜炎中LOX-1和TLR4可以相互作用,并且通过调节活性氧(ROS)的产生调节炎症。方法:用具有活性的烟曲霉菌菌丝感染C57BL/6小鼠角膜,制作小鼠烟曲霉菌性角膜炎模型,用灭活的烟曲霉菌菌丝刺激从C57BL/6小鼠腹腔提取的中性粒细胞。采用LOX-1中和抗体、Poly(I)(LOX-1抑制剂)及CLI-095(TLR4抑制剂)预处理C57BL/6小鼠角膜后再用有活性的烟曲霉菌菌丝感染。采用real-time RT-PCR、western blot方法检测正常和烟曲霉菌感染角膜中的LOX-1、TLR4和IL-1β的mRNA和蛋白水平的表达,以及检测正常和烟曲霉菌菌丝刺激的中性粒细胞中的LOX-1、TLR4和IL-1β的mRNA表达。采用流式细胞术检测从C57BL/6小鼠腹腔提取的中性粒细胞中ROS的产生情况,观察烟曲霉菌菌丝刺激中性粒细胞后ROS的生成变化。用LOX-1中和抗体、Poly(I)(LOX-1抑制剂)及CLI-095(TLR4抑制剂)预处理从C57BL/6小鼠腹腔提取的中性粒细胞后再用灭活的烟曲霉菌菌丝刺激,再采用流式细胞术检测预处理后中性粒细胞中ROS的生成变化。结果:烟曲霉菌菌丝感染C57BL/6小鼠角膜后LOX-1、TLR4和IL-1β的mRNA和蛋白表达水平增高。当采用LOX-1中和抗体预处理C57BL/6小鼠角膜阻断LOX-1后,再用烟曲霉菌菌丝感染,TLR4和IL-1β的mRNA表达水平都降低。为进一步验证,采用LOX-1抑制剂Poly(I)预处理C57BL/6小鼠角膜阻断LOX-1后,再用烟曲霉菌菌丝感染,TLR4和IL-1β的mRNA表达水平也都降低。进一步检测蛋白水平发现,采用LOX-1中和抗体和LOX-1抑制剂Poly(I)预处理C57BL/6小鼠角膜阻断LOX-1后,再用烟曲霉菌菌丝感染,TLR4和IL-1β的蛋白表达水平都降低。当采用CLI-095预处理C57BL/6小鼠角膜阻断TLR4后,再用烟曲霉菌菌丝感染时,LOX-1和IL-1β的mRNA表达水平都降低。进一步检测蛋白水平发现,采用CLI-095预处理C57BL/6小鼠角膜阻断TLR4后,再用烟曲霉菌菌丝感染时,LOX-1和IL-1β的蛋白表达水平也都降低。与正常组相比,灭活的烟曲霉菌菌丝刺激C57BL/6小鼠腹腔提取的中性粒细胞后,LOX-1、TLR4和IL-1β的mRNA表达水平升高,并且在烟曲霉菌刺激后8小时达到高峰,随后表达降低。流式细胞术检测结果显示:与正常组相比,灭活的烟曲霉菌菌丝刺激C57BL/6小鼠腹腔提取的中性粒细胞后,ROS产生增多。采用LOX-1中和抗体、Poly(I)预处理C57BL/6小鼠腹腔中性粒细胞阻断LOX-1后,再用灭活的烟曲霉菌菌丝刺激时,ROS的产生减少。采用CLI-095预处理C57BL/6小鼠中性粒细胞阻断TLR4后,再用灭活的烟曲霉菌菌丝刺激时,ROS的产生减少。结论:C57BL/6小鼠烟曲霉菌性角膜炎中LOX-1、TLR4和IL-1β的表达升高。抑制LOX-1可以降低TLR4和IL-1β的表达,抑制TLR4可以降低LOX-1和IL-1β的表达,说明烟曲霉菌感染C57BL/6小鼠角膜的过程中,LOX-1和TLR4可以相互作用,促进炎症。烟曲霉菌感染后中性粒细胞中ROS的产生增多,抑制LOX-1或抑制TLR4都可以减少ROS的生成。在烟曲霉菌性角膜炎中LOX-1和TLR4可以相互作用并且可以通过调节ROS的产生来调节炎症。
[Abstract]:Objective: To investigate the relationship between LOX-1 and TLR4 in Aspergillus fumigatus keratitis, it is proved that LOX-1 and TLR4 can interact in the Aspergillus fumigatus keratitis and regulate the inflammation by regulating the production of reactive oxygen species (ROS). Methods: using the active mycelium of Aspergillus fumigatus to infect the cornea of C57BL /6 in mice and make the mold of Aspergillus fumigatus in mice. Type, using inactivated Aspergillus fumigatus mycelium to stimulate neutrophils extracted from C57BL/6 mouse abdominal cavity. Using LOX-1 neutralization antibody, Poly (I) (LOX-1 inhibitor) and CLI-095 (TLR4 inhibitor) pretreated C57BL/6 mice cornea after treatment with active infection of Aspergillus fumigatus mycelium. Real-time RT-PCR, Western blot methods were used to detect normal and Aspergillus fumigatus The expression of mRNA and protein levels of LOX-1, TLR4 and IL-1 beta in the cornea, and the detection of mRNA expression of LOX-1, TLR4 and IL-1 beta in neutrophils stimulated by normal and Aspergillus fumigatus. Flow cytometry was used to detect the occurrence of ROS in the neutrophils extracted from the peritoneal cavity of the C57BL/6 mice and to observe the stimulation of Aspergillus fumigatus mycelium. Changes in the generation of ROS after neutrophils. Using LOX-1 neutralizing antibody, Poly (I) (LOX-1 inhibitor) and CLI-095 (TLR4 inhibitor) preconditioning from the neutrophils extracted from the peritoneal cavity of C57BL/6 mice, then using inactivated Aspergillus fumigatus mycelium, and then using flow cytometry to detect the formation and change of ROS in neutrophils after pretreatment. Results: Aspergillus fumigatus The level of mRNA and protein expression of LOX-1, TLR4 and IL-1 beta in the cornea of C57BL/6 mice increased after the infection of mycelium in the cornea of mice. When the LOX-1 neutralized antibody was pretreated with the cornea of the C57BL/6 mice, the LOX-1 was blocked, and the infection of Aspergillus fumigatus mycelia, the mRNA expression level of TLR4 and IL-1 beta was reduced. After blocking LOX-1 in the cornea, the mRNA expression level of TLR4 and IL-1 beta was also reduced with the infection of Aspergillus fumigatus mycelium. Further detection of protein levels found that LOX-1 neutralization antibody and LOX-1 inhibitor Poly (I) were used to pretreat C57BL/6 mouse cornea blocking LOX-1, and then Aspergillus fumigatus mycelium infection, TLR4 and IL-1 beta protein expression levels were reduced. When the C57BL/6 mice were pretreated with CLI-095 to block the TLR4, the mRNA expression level of LOX-1 and IL-1 beta was reduced with the infection of Aspergillus fumigatus mycelium. Further detection of protein levels found that CLI-095 pretreated C57BL/6 mice cornea to block TLR4 and then the protein expression level of LOX-1 and IL-1 beta was also in the case of Aspergillus fumigatus infection. Decrease. Compared with the normal group, the inactivated Aspergillus fumigatus mycelium stimulated the mRNA expression level of LOX-1, TLR4 and IL-1 beta in C57BL/6 mice, and reached the peak at 8 hours after the stimulation of Aspergillus fumigatus and then decreased. The flow cytometry showed that the inactivated Aspergillus fumigatus was compared with the normal group. After stimulating the neutrophils extracted from the peritoneal cavity of C57BL/6 mice, the production of ROS increased. Using LOX-1 neutralization antibody, Poly (I) pretreated C57BL/6 mouse peritoneal neutrophils to block LOX-1, the production of ROS decreased with inactivated Aspergillus fumigatus hypha stimulation. When the mycelium of Aspergillus fumigatus was stimulated, the production of ROS decreased. Conclusion: the expression of LOX-1, TLR4 and IL-1 beta in C57BL/6 mice increased. The inhibition of LOX-1 could reduce the expression of TLR4 and IL-1 beta, and the inhibition of TLR4 could reduce the expression of LOX-1 and IL-1 beta. Interaction, promoting inflammation. The increase of ROS in neutrophils after Aspergillus fumigatus infection, inhibition of LOX-1 or inhibition of TLR4 can reduce the formation of ROS. In the Aspergillus fumigatus keratitis, LOX-1 and TLR4 can interact and can regulate the inflammation by regulating the production of ROS.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R772.21

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