本文選題：煙曲霉菌 + 角膜炎��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的:為了探討煙曲霉菌性角膜炎中LOX-1和TLR4的關(guān)系,證明在煙曲霉菌性角膜炎中LOX-1和TLR4可以相互作用,并且通過調(diào)節(jié)活性氧(ROS)的產(chǎn)生調(diào)節(jié)炎癥。方法:用具有活性的煙曲霉菌菌絲感染C57BL/6小鼠角膜,制作小鼠煙曲霉菌性角膜炎模型,用滅活的煙曲霉菌菌絲刺激從C57BL/6小鼠腹腔提取的中性粒細(xì)胞。采用LOX-1中和抗體、Poly(I)(LOX-1抑制劑)及CLI-095(TLR4抑制劑)預(yù)處理C57BL/6小鼠角膜后再用有活性的煙曲霉菌菌絲感染。采用real-time RT-PCR、western blot方法檢測正常和煙曲霉菌感染角膜中的LOX-1、TLR4和IL-1β的mRNA和蛋白水平的表達,以及檢測正常和煙曲霉菌菌絲刺激的中性粒細(xì)胞中的LOX-1、TLR4和IL-1β的mRNA表達。采用流式細(xì)胞術(shù)檢測從C57BL/6小鼠腹腔提取的中性粒細(xì)胞中ROS的產(chǎn)生情況,觀察煙曲霉菌菌絲刺激中性粒細(xì)胞后ROS的生成變化。用LOX-1中和抗體、Poly(I)(LOX-1抑制劑)及CLI-095(TLR4抑制劑)預(yù)處理從C57BL/6小鼠腹腔提取的中性粒細(xì)胞后再用滅活的煙曲霉菌菌絲刺激,再采用流式細(xì)胞術(shù)檢測預(yù)處理后中性粒細(xì)胞中ROS的生成變化。結(jié)果:煙曲霉菌菌絲感染C57BL/6小鼠角膜后LOX-1、TLR4和IL-1β的mRNA和蛋白表達水平增高。當(dāng)采用LOX-1中和抗體預(yù)處理C57BL/6小鼠角膜阻斷LOX-1后,再用煙曲霉菌菌絲感染,TLR4和IL-1β的mRNA表達水平都降低。為進一步驗證,采用LOX-1抑制劑Poly(I)預(yù)處理C57BL/6小鼠角膜阻斷LOX-1后,再用煙曲霉菌菌絲感染,TLR4和IL-1β的mRNA表達水平也都降低。進一步檢測蛋白水平發(fā)現(xiàn),采用LOX-1中和抗體和LOX-1抑制劑Poly(I)預(yù)處理C57BL/6小鼠角膜阻斷LOX-1后,再用煙曲霉菌菌絲感染,TLR4和IL-1β的蛋白表達水平都降低。當(dāng)采用CLI-095預(yù)處理C57BL/6小鼠角膜阻斷TLR4后,再用煙曲霉菌菌絲感染時,LOX-1和IL-1β的mRNA表達水平都降低。進一步檢測蛋白水平發(fā)現(xiàn),采用CLI-095預(yù)處理C57BL/6小鼠角膜阻斷TLR4后,再用煙曲霉菌菌絲感染時,LOX-1和IL-1β的蛋白表達水平也都降低。與正常組相比,滅活的煙曲霉菌菌絲刺激C57BL/6小鼠腹腔提取的中性粒細(xì)胞后,LOX-1、TLR4和IL-1β的mRNA表達水平升高,并且在煙曲霉菌刺激后8小時達到高峰,隨后表達降低。流式細(xì)胞術(shù)檢測結(jié)果顯示:與正常組相比,滅活的煙曲霉菌菌絲刺激C57BL/6小鼠腹腔提取的中性粒細(xì)胞后,ROS產(chǎn)生增多。采用LOX-1中和抗體、Poly(I)預(yù)處理C57BL/6小鼠腹腔中性粒細(xì)胞阻斷LOX-1后,再用滅活的煙曲霉菌菌絲刺激時,ROS的產(chǎn)生減少。采用CLI-095預(yù)處理C57BL/6小鼠中性粒細(xì)胞阻斷TLR4后,再用滅活的煙曲霉菌菌絲刺激時,ROS的產(chǎn)生減少。結(jié)論:C57BL/6小鼠煙曲霉菌性角膜炎中LOX-1、TLR4和IL-1β的表達升高。抑制LOX-1可以降低TLR4和IL-1β的表達,抑制TLR4可以降低LOX-1和IL-1β的表達,說明煙曲霉菌感染C57BL/6小鼠角膜的過程中,LOX-1和TLR4可以相互作用,促進炎癥。煙曲霉菌感染后中性粒細(xì)胞中ROS的產(chǎn)生增多,抑制LOX-1或抑制TLR4都可以減少ROS的生成。在煙曲霉菌性角膜炎中LOX-1和TLR4可以相互作用并且可以通過調(diào)節(jié)ROS的產(chǎn)生來調(diào)節(jié)炎癥。
[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.

【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】：2017
【分類號】：R772.21

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