肠急性损伤条件下肠上皮间淋巴细胞对肠上皮细胞作用及机制研究
发布时间：2018-08-13 09:01
【摘要】：背景与目的：肠黏膜屏障是机体抵御肠腔病原体入侵的重要屏障。研究表明：缺血再灌注(ischemia/reperfusion, I/R)、应激、放射性损伤等急性病理刺激下,均会发生肠黏膜屏障功能受损,导致肠源性感染,加重原发疾病,最终引起多器官功能衰竭。尽管目前针对肠黏膜屏障损伤与保护等领域做了大量研究工作,但肠黏膜屏障中涉及免疫调控与免疫反应的机制仍未阐明。针对当前肠黏膜屏障急性损伤后继发的免疫功能紊乱甚至免疫反应介导的“二次损伤”等复杂问题,迫切要求我们阐明肠黏膜免疫功能紊乱及调节的关键机制,为急性病理条件下肠黏膜屏障保护提供新策略。作为肠黏膜免疫系统的重要一员,肠上皮间淋巴细胞(Intraepithelial lymphocyte, IELs)是定植于肠上皮细胞间的一类独特的淋巴细胞群。IELs由多种亚群构成,主要包括TCRγδ+T细胞、TCRαβ+T细胞。TCRαβ+IELs又可分为TCRαβ+CD8aa+IELs、 TCRαβ+CD8αβ+IELs和TCRαβ+CD4+IELs三个亚群；TCRγδ+几乎全部为CD8αα+。IELs的多亚群构成,也提示了它们来源的多样性。因此,IELs来源多样,功能复杂,已成为近年来肠道免疫研究的热点。在生理情况下,IELs分泌细胞因子(如KGF)、抗菌肽等,维持上皮细胞生长及抵御病原体入侵；病理情况下,IELs快速做出应答,从半活化状态迅速进入活化状态,分泌细胞因子参与炎症反应,同时这些细胞因子也可能介导对肠黏膜的损伤,导致屏障功能下降。前期研究发现,小肠I/R可以直接损伤肠上皮细胞以及细胞间连接。例如：肠黏膜绒毛的明显断裂、脱落和肠道上皮细胞的凋亡增加、坏死；维持IELs在上皮细胞间定植的紧密连接蛋白(Claudin-1和Occludin)结构破坏；肠黏膜通透性增加。我们在病人缺血的肠道标本中发现,IL-7表达增多。IL-7作为肠上皮细胞分泌的、刺激淋巴细胞发育的细胞因子,在维持IELs表型和功能方面发挥了重要作用。在我们课题组前期构建的IL-7肠道过表达小鼠中发现,IELs表现为Thl型细胞因子分泌增多,活化标志CD69+表达上调。提示：IL-7的上调,介导淋巴细胞过度活化,参与炎症反应。IELs无论空间距离和功能调控都与肠上皮细胞有着紧密联系。因此,我们推测I/R可导致IELs的过度活化,同时,活化后的IELs介导了对肠黏膜屏障的损伤。我们在本论文中重点探讨小肠I/R条件下IELs的功能变化,以及其对上皮屏障产生的影响。同时,我们也注意到,急性放射性损伤可以导致上皮细胞大量死亡,由上皮细胞分泌的IL-7是否会受到影响。那么,这种情况下,IELs又将发生什么变化呢?我们针对这一问题也将进行初步的探讨。因此本研究运用流式细胞分选,IELs上清与上皮细胞共培养、免疫组化、蛋白印迹、ELISA等技术,探讨I/R和急性放射性损伤条件下(以I/R为重点),IELs亚型及功能的变化规律,以及IELs功能改变后对肠黏膜屏障损伤的相关机制。其中包括：明确对上皮屏障造成损伤的IELs源性细胞因子,该细胞因子对紧密连接蛋白如Claudin-1、Occludin等的表达及结构变化情况；并通过检测跨上皮电阻抗(TER),确定IELs对屏障功能的调控作用；最后人为干预该细胞因子,评估肠屏障损伤的改善情况。为进一步阐明肠急性损伤条件下IELs的变化规律及其调控肠黏膜屏障的作用机制提供理论依据,为肠黏膜屏障损伤干预提供新靶点。方法：建立小鼠急性小肠I/R模型及急性放射性损伤模型,运用HE染色,免疫组化,Ussing Chamer, Western Blot等技术了解急性损伤情况下肠黏膜的改变情况。围绕肠I/R模型,运用流式细胞技术、免疫组化、定量PCR技术,阐明I/R条件下IELs表型和功能变化规律。运用流式细胞分选IELs,结合IELs上清与上皮细胞共培养模型,明确IELs源性细胞因子TNF-a对上皮细胞的作用,以及细胞因子对上皮间紧密连接蛋白表达及结构的影响。使用抗TNF-a抗体术前干预,通过跨上皮电阻抗(TER)及组织损伤评分的技术手段,明确IELs源细胞因子在肠黏膜屏障损伤中发挥了关键作用。结果：肠I/R条件下,小肠黏膜正常结构受损,绒毛断裂,上皮下间隙增大,上皮脱落,固有层细胞成分增多,肠黏膜通透性增大。肠I/R后肠黏膜紧密连接蛋白(Claudin-1, Occludin)表达下降,Occludin在上皮细胞顶端正常结构遭到破坏。急性肠I/R对IELs数量没有影响,但导致IELs与上皮细胞的正常连接消失。肠I/R条件下,上皮细胞分泌IL-7增多,IELs亚群CD8ap+, CD4+和TCRa(3+的百分比增高,CD8+, CD4+ IELs凋亡先后开始启动。并且CD8+, CD4+ IELs活化显著增加,TNF-α、IL-10mRNA拷贝数增多,KGF减少,IFN-y没有明显变化。I/R处理后的IELs上清与上皮细胞共培养后,发现上皮细胞Claudin-1、Occludin表达下降,Occludin结构完整性破坏,上皮屏障通透性增高,在I/R处理后的IELs上清中加入TNF-a抗体,可以逆转上述改变。I/R处理后,CD45+IELs胞内合成以及分泌的TNF-a增多,用IL-7刺激体外培养的IELs后,TNF-a分泌明显增多。抗TNF-a治疗可以显著缓解I/R小鼠肠黏膜损伤。急性放射性肠损伤直接导致肠黏膜结构破坏严重,绒毛形态不能正常维持,凋亡细胞增多,肠道IL-7表达降低,肠道IELs细胞数量72h后减少非常明显。给予外源性IL-7处理,急性放射性肠损伤导致的IELs数量降低得到改善。结论：急性I/R条件下：IELs受到影响,表现为与上皮细胞间连接结构破坏,IELs亚群比例失调；IELs活化增加,其分泌的TNF-a介导了对上皮细胞的损伤以及上皮间紧密连接蛋白正常结构的破坏。抗TNF-a治疗可以显著改善肠黏膜损伤。急性放射性肠损伤条件下：肠黏膜结构严重破坏,绒毛形态不能维持,凋亡细胞增多,肠道上皮表达IL-7降低是IELs细胞数量减少的重要因素之一,给予外源性IL-7治疗可以缓解IELs数量降低,改善放射损伤后肠黏膜免疫功能。
[Abstract]:BACKGROUND & OBJECTIVE: The intestinal mucosal barrier is an important barrier for the body to resist the invasion of enteric pathogens. Studies have shown that acute pathological stimuli such as ischemia/reperfusion (I/R), stress and radiation damage can lead to impaired intestinal mucosal barrier function, intestinal infection, aggravation of primary diseases and eventual multi-organ function. Fatigue. Although a lot of research work has been done on the damage and protection of intestinal mucosal barrier, the mechanisms involved in immune regulation and immune response in intestinal mucosal barrier remain unclear. As an important member of the intestinal mucosal immune system, intestinal interepithelial lymphocyte (IELs) is a unique group of lymphocytes colonized between intestinal epithelial cells. IELs are composed of many subgroups, mainly including TCR gamma delta + T cells, TCR alpha beta + T cells. TCR alpha beta + IELs can be divided into TCR alpha beta + CD8aa + IELs, TCR alpha beta + CD8 alpha beta + IELs and TCR alpha beta + CD4 + IELs. TCR gamma delta + is almost all composed of CD8 alpha +. IELs, indicating the diversity of their sources. In physiological conditions, IELs secrete cytokines (such as KGF) and antimicrobial peptides to maintain epithelial cell growth and resist pathogen invasion; in pathological conditions, IELs respond rapidly, from semi-activated state to activated state, secreting cytokines to participate in inflammatory reaction, and these cells at the same time. Previous studies have shown that small intestinal I/R can directly damage intestinal epithelial cells and intercellular junctions. For example, intestinal mucosal villi are ruptured, exfoliation and intestinal epithelial cells apoptosis and necrosis; IELs are maintained in tight junction proteins (CTPs) between epithelial cells. Claudin-1 and Occludin were destroyed and intestinal mucosal permeability was increased. We found that IL-7 expression was increased in ischemic intestinal specimens. IL-7, a cytokine secreted by intestinal epithelial cells and stimulating lymphocyte development, played an important role in maintaining the phenotype and function of IELs. IL-7 intestinal tract, which was constructed earlier in our research group, played an important role in maintaining the phenotype and function of IELs. In overexpressed mice, IELs showed increased secretion of Thl-type cytokines and up-regulated expression of CD69 +. These results suggest that the up-regulation of IL-7 may mediate lymphocyte over-activation and participate in inflammatory reaction. IELs are closely related to intestinal epithelial cells regardless of spatial distance and functional regulation. In this study, we focused on the functional changes of IELs under small intestinal I/R and their effects on the epithelial barrier. At the same time, we also noted that acute radiation injury can cause a large number of epithelial cell death, and whether IL-7 secreted by epithelial cells will be affected. So, what changes will happen to IELs in this case? We will also make a preliminary study on this issue. Therefore, this study used flow cytometry, IELs supernatant co-culture with epithelial cells, immunohistochemistry, Western blotting, ELISA and other techniques to explore the conditions of I/R and acute radiation injury (with emphasis on I/R), IELs subgroup. These include: to identify the IELs-derived cytokines that cause damage to the epithelial barrier, the expression and structural changes of tight junction proteins such as Claudin-1 and Occludin, and to detect transepithelial electrical impedance (TER). To determine the regulatory effect of IELs on intestinal barrier function, and finally to evaluate the improvement of intestinal barrier injury by interfering with this cytokine. HE staining, immunohistochemistry, Ussing Chamer, Western Blot and other techniques were used to study the changes of intestinal mucosa in acute intestinal I/R model and acute radiation injury model in mice. The effect of IELs-derived cytokine TNF-a on epithelial cells and the effect of cytokines on the expression and structure of tight junction protein were studied by flow cytometry and co-culture model of IELs supernatant and epithelial cells. Results: Under intestinal I/R condition, the normal structure of intestinal mucosa was damaged, the villi were broken, the subepithelial space was enlarged, the epithelium was exfoliated, the cell components in lamina propria were increased, and the intestinal mucosal permeability was increased. Acute intestinal I/R did not affect the number of IELs, but led to the disappearance of normal junction between IELs and epithelial cells. Under intestinal I/R conditions, the secretion of IL-7 by epithelial cells increased, the percentage of IELs CD8ap +, CD4 + and TCRa (3 +) increased, and the apoptosis of CD8 +, CD4 + IELs began successively. CD4+IELs activation increased significantly, TNF-a, IL-10 mRNA copies increased, KGF decreased, IFN-y did not change significantly. After co-culture with epithelial cells, the expression of Claudin-1, Occludin decreased, the structural integrity of Occludin destroyed, and the permeability of epithelial barrier increased. TNF-a was added to the supernatant of IELs after I/R treatment. Anti-TNF-a treatment can significantly alleviate the intestinal mucosal injury in I/R mice. Acute radiation-induced intestinal injury can lead to serious damage of intestinal mucosal structure and abnormal chorionic villi morphology. After treatment with exogenous IL-7, the number of IELs decreased significantly. CONCLUSION: Under acute I/R condition, IELs were impaired, and the ratio of IELs to epithelial cells was imbalance. Anti-TNF-a therapy can significantly improve intestinal mucosal injury. Under acute radiation-induced intestinal injury, intestinal mucosal structure is severely damaged, villi morphology is not maintained, apoptotic cells are increased, and intestinal epithelial expression is increased. The decrease of IL-7 is one of the important factors for the decrease of the number of IELs cells. Exogenous IL-7 therapy can relieve the decrease of the number of IELs and improve the intestinal mucosal immune function after radiation injury.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R574
[Abstract]:BACKGROUND & OBJECTIVE: The intestinal mucosal barrier is an important barrier for the body to resist the invasion of enteric pathogens. Studies have shown that acute pathological stimuli such as ischemia/reperfusion (I/R), stress and radiation damage can lead to impaired intestinal mucosal barrier function, intestinal infection, aggravation of primary diseases and eventual multi-organ function. Fatigue. Although a lot of research work has been done on the damage and protection of intestinal mucosal barrier, the mechanisms involved in immune regulation and immune response in intestinal mucosal barrier remain unclear. As an important member of the intestinal mucosal immune system, intestinal interepithelial lymphocyte (IELs) is a unique group of lymphocytes colonized between intestinal epithelial cells. IELs are composed of many subgroups, mainly including TCR gamma delta + T cells, TCR alpha beta + T cells. TCR alpha beta + IELs can be divided into TCR alpha beta + CD8aa + IELs, TCR alpha beta + CD8 alpha beta + IELs and TCR alpha beta + CD4 + IELs. TCR gamma delta + is almost all composed of CD8 alpha +. IELs, indicating the diversity of their sources. In physiological conditions, IELs secrete cytokines (such as KGF) and antimicrobial peptides to maintain epithelial cell growth and resist pathogen invasion; in pathological conditions, IELs respond rapidly, from semi-activated state to activated state, secreting cytokines to participate in inflammatory reaction, and these cells at the same time. Previous studies have shown that small intestinal I/R can directly damage intestinal epithelial cells and intercellular junctions. For example, intestinal mucosal villi are ruptured, exfoliation and intestinal epithelial cells apoptosis and necrosis; IELs are maintained in tight junction proteins (CTPs) between epithelial cells. Claudin-1 and Occludin were destroyed and intestinal mucosal permeability was increased. We found that IL-7 expression was increased in ischemic intestinal specimens. IL-7, a cytokine secreted by intestinal epithelial cells and stimulating lymphocyte development, played an important role in maintaining the phenotype and function of IELs. IL-7 intestinal tract, which was constructed earlier in our research group, played an important role in maintaining the phenotype and function of IELs. In overexpressed mice, IELs showed increased secretion of Thl-type cytokines and up-regulated expression of CD69 +. These results suggest that the up-regulation of IL-7 may mediate lymphocyte over-activation and participate in inflammatory reaction. IELs are closely related to intestinal epithelial cells regardless of spatial distance and functional regulation. In this study, we focused on the functional changes of IELs under small intestinal I/R and their effects on the epithelial barrier. At the same time, we also noted that acute radiation injury can cause a large number of epithelial cell death, and whether IL-7 secreted by epithelial cells will be affected. So, what changes will happen to IELs in this case? We will also make a preliminary study on this issue. Therefore, this study used flow cytometry, IELs supernatant co-culture with epithelial cells, immunohistochemistry, Western blotting, ELISA and other techniques to explore the conditions of I/R and acute radiation injury (with emphasis on I/R), IELs subgroup. These include: to identify the IELs-derived cytokines that cause damage to the epithelial barrier, the expression and structural changes of tight junction proteins such as Claudin-1 and Occludin, and to detect transepithelial electrical impedance (TER). To determine the regulatory effect of IELs on intestinal barrier function, and finally to evaluate the improvement of intestinal barrier injury by interfering with this cytokine. HE staining, immunohistochemistry, Ussing Chamer, Western Blot and other techniques were used to study the changes of intestinal mucosa in acute intestinal I/R model and acute radiation injury model in mice. The effect of IELs-derived cytokine TNF-a on epithelial cells and the effect of cytokines on the expression and structure of tight junction protein were studied by flow cytometry and co-culture model of IELs supernatant and epithelial cells. Results: Under intestinal I/R condition, the normal structure of intestinal mucosa was damaged, the villi were broken, the subepithelial space was enlarged, the epithelium was exfoliated, the cell components in lamina propria were increased, and the intestinal mucosal permeability was increased. Acute intestinal I/R did not affect the number of IELs, but led to the disappearance of normal junction between IELs and epithelial cells. Under intestinal I/R conditions, the secretion of IL-7 by epithelial cells increased, the percentage of IELs CD8ap +, CD4 + and TCRa (3 +) increased, and the apoptosis of CD8 +, CD4 + IELs began successively. CD4+IELs activation increased significantly, TNF-a, IL-10 mRNA copies increased, KGF decreased, IFN-y did not change significantly. After co-culture with epithelial cells, the expression of Claudin-1, Occludin decreased, the structural integrity of Occludin destroyed, and the permeability of epithelial barrier increased. TNF-a was added to the supernatant of IELs after I/R treatment. Anti-TNF-a treatment can significantly alleviate the intestinal mucosal injury in I/R mice. Acute radiation-induced intestinal injury can lead to serious damage of intestinal mucosal structure and abnormal chorionic villi morphology. After treatment with exogenous IL-7, the number of IELs decreased significantly. CONCLUSION: Under acute I/R condition, IELs were impaired, and the ratio of IELs to epithelial cells was imbalance. Anti-TNF-a therapy can significantly improve intestinal mucosal injury. Under acute radiation-induced intestinal injury, intestinal mucosal structure is severely damaged, villi morphology is not maintained, apoptotic cells are increased, and intestinal epithelial expression is increased. The decrease of IL-7 is one of the important factors for the decrease of the number of IELs cells. Exogenous IL-7 therapy can relieve the decrease of the number of IELs and improve the intestinal mucosal immune function after radiation injury.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R574
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