小肠上皮内淋巴细胞在抵抗鼠伤寒沙门氏菌感染中的作用及机制探讨

发布时间：2018-01-04 09:31

本文关键词：小肠上皮内淋巴细胞在抵抗鼠伤寒沙门氏菌感染中的作用及机制探讨　出处：《山东大学》2011年硕士论文　论文类型：学位论文

更多相关文章： 上皮内淋巴细胞 伤寒 NKG2D Qa-1b

【摘要】：目的肠道免疫系统对于维持肠道粘膜的稳态发挥着重要作用,它既要保护机体免受病原菌的入侵,又要对肠道中的共生菌保持耐受,其区分共生菌群和致病微生物的机制至今仍未得到阐明。肠道上皮内淋巴细胞(iIELs)镶嵌在肠道上皮细胞间,对粘膜免疫应答的调节起着重要作用。iIELs被认为是一群高度活化的大颗粒淋巴细胞,可以迅速地对早期感染产生免疫应答。本研究选用一种肠道致病菌鼠伤寒沙门氏菌,观察小鼠经口感染后其iIELs不同亚群的表型变化,并以肠道共生菌之一大肠杆菌为对照,探究在致病菌感染过程中发挥主要抵抗作用的iIELs亚群,并探讨其抗感染的机制,以便于进一步研究在肠道感染过程中免疫应答及免疫病理的发生机制,进而为肠道感染及肿瘤等疾病的治疗提供新的策略和思路。方法 1.小鼠灌胃感染鼠伤寒沙门氏菌,建立肠道感染模型,HE染色检测伤寒感染后小肠粘膜形态变化。 2.MTT法检测伤寒感染后iIELs对鼠T淋巴瘤细胞系YAC-1、鼠结直肠癌细胞系MCA-38及鼠小肠上皮细胞(IECs)杀伤能力的变化。 3. RT-PCR检测伤寒感染后iIELs杀伤相关分子基因水平的表达变化；Real-TimePCR检测伤寒感染后小肠上皮细胞Qa-1及NKG2D相关配体基因水平表达的变化。 4.小鼠体内阻断NKG2D后检测小肠组织中伤寒菌的负载量。 5.流式细胞术检测伤寒感染后iIELs各亚群及IECs的表型变化。 6.免疫磁珠法分选CD8+TCRγδ+iIELs亚群,检测伤寒感染后该亚群的表型和杀伤功能的变化。 7.小鼠体内清除γδT细胞后检测肝、脾、小肠及派氏结中伤寒菌的负载量。结果 1.鼠伤寒沙门氏菌经口感染可引起小鼠肠炎； 2.鼠伤寒沙门氏菌感染时CD8+TCRγδ+ilELs亚群的比例增加,iIELs对肿瘤细胞及感染的肠上皮细胞杀伤活性增强； 3.鼠伤寒沙门氏菌感染可致CD8+TCRγδ+iIELs亚群表面NKG2D的表达增强； 4.鼠伤寒沙门氏菌感染可致IECs表面MULT1表达增强； 5.体内阻断NKG2D增加了伤寒菌在小肠中的负载量； 6.体内清除γδT细胞加剧了伤寒菌对机体的侵袭； 7.鼠伤寒沙门氏菌感染降低了IECs表面Qa-1b的表达水平； 8.小鼠iIELs对共生菌经口感染保持耐受状态； 9.Nodl可能参与IECs对伤寒菌的识别。结论 1. iIELs,尤其是CD8+TCRγδ+iIELs亚群,在小肠抵抗鼠伤寒沙门氏菌感染中发挥重要作用； 2.伤寒感染时CD8+TCRγδ+iIELs亚群表面NKG2D表达增强,肠上皮细胞NKG2D配体MULT-1表达增强,而抑制性配体Qa-1b表达下降,导致活化性信号和抑制性信号平衡的失调； 3.NKG2D及其配体的相互识别参与了CD8+TCRy8+iIELs亚群对伤寒感染的肠上皮细胞的清除； 4.肠上皮细胞Qa-1 b高表达可能是其免受iIELs攻击,保持对肠道共生菌耐受的机制之一。
[Abstract]:Purpose The intestinal immune system plays an important role in maintaining the homeostasis of intestinal mucosa. It not only protects the body from the invasion of pathogens, but also maintains tolerance to symbiotic bacteria in the intestinal tract. The mechanism of differentiation between symbiotic flora and pathogenic microorganism has not been elucidated. Intestinal intraepithelial lymphocytes (iIELs) are embedded in intestinal epithelial cells. The regulation of mucosal immune response plays an important role. IIELs are considered to be a group of highly activated large granular lymphocytes which can react quickly to early infection. In this study, the phenotypic changes of different subgroups of iIELs in mice after oral infection were observed, and Escherichia coli, one of the enteric symbiotic bacteria, was used as control. To explore the iIELs subgroup which plays a major role in the process of pathogenic bacteria infection and to explore its anti-infection mechanism in order to further study the immune response and the pathogenesis of immunopathology in the process of intestinal infection. It provides new strategies and ideas for the treatment of intestinal infection and tumor. Method 1. Mice were infected with Salmonella typhimurium. The intestinal infection model was established to detect the changes of intestinal mucosal morphology after typhoid infection by HE staining. 2. MTT assay was used to detect the effect of iIELs on T lymphoma cell line YAC-1 after typhoid infection. Changes of cytotoxicity of murine colorectal cancer cell line MCA-38 and intestinal epithelial cells (IECs). 3. RT-PCR was used to detect the expression of iIELs killer related gene after typhoid infection. Real-TimePCR was used to detect the expression of Qa-1 and NKG2D related ligand genes in intestinal epithelial cells after typhoid infection. 4. After blocking NKG2D in mice, the loading amount of typhoid bacteria in small intestine was detected. 5. The phenotypic changes of iIELs subsets and IECs after typhoid infection were detected by flow cytometry. 6. The CD8 TCR 纬未 iIELs subsets were separated by immunomagnetic beads, and the phenotype and killing function of the subgroups were detected after typhoid infection. 7. After removing 纬未 T cells in mice, the loading amount of typhoid bacteria in liver, spleen, small intestine and Pak's knot was detected. Results 1. The oral infection of Salmonella typhimurium could cause enteritis in mice. 2. The proportion of CD8 TCR 纬未 ilELs subsets increased with the infection of Salmonella typhimurium. 3.The expression of NKG2D on the surface of CD8 TCR 纬未 iIELs subgroup was enhanced by Salmonella typhimurium infection. 4. Salmonella typhimurium infection increased the expression of MULT1 on the surface of IECs. 5. Blocking NKG2D in vivo increased the amount of typhoid bacteria loading in the small intestine. 6. Removal of 纬未 T cells in vivo aggravated the invasion of typhoid. 7. Salmonella typhimurium infection decreased the expression of Qa-1b on the surface of IECs. 8. Mouse iIELs maintained tolerance to symbiotic bacteria by oral infection. 9. Nodl may be involved in the identification of typhoid by IECs. Conclusion 1. IIELs, especially CD8 TCR 纬未 iIELs subsets, play an important role in intestinal resistance to Salmonella typhimurium infection. 2. The expression of NKG2D on the surface of CD8 TCR 纬未 iIELs subgroup and MULT-1 expression of NKG2D ligand in intestinal epithelial cells were increased during typhoid infection. However, the decrease of Qa-1b expression of inhibitory ligand resulted in the imbalance of activation signal and inhibitory signal balance. 3. The recognition of NKG2D and its ligand involved in the clearance of typhoid infected intestinal epithelial cells by CD8 TCRy8 iIELs subsets. 4. The high expression of Qa-1 b in intestinal epithelial cells may be one of the mechanisms of its tolerance to intestinal symbiotic bacteria from iIELs attack.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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