腸急性損傷條件下腸上皮間淋巴細胞對腸上皮細胞作用及機制研究
發(fā)布時間：2018-08-13 09:01
【摘要】：背景與目的：腸黏膜屏障是機體抵御腸腔病原體入侵的重要屏障。研究表明：缺血再灌注(ischemia/reperfusion, I/R)、應激、放射性損傷等急性病理刺激下,均會發(fā)生腸黏膜屏障功能受損,導致腸源性感染,加重原發(fā)疾病,最終引起多器官功能衰竭。盡管目前針對腸黏膜屏障損傷與保護等領域做了大量研究工作,但腸黏膜屏障中涉及免疫調控與免疫反應的機制仍未闡明。針對當前腸黏膜屏障急性損傷后繼發(fā)的免疫功能紊亂甚至免疫反應介導的“二次損傷”等復雜問題,迫切要求我們闡明腸黏膜免疫功能紊亂及調節(jié)的關鍵機制,為急性病理條件下腸黏膜屏障保護提供新策略。作為腸黏膜免疫系統(tǒng)的重要一員,腸上皮間淋巴細胞(Intraepithelial lymphocyte, IELs)是定植于腸上皮細胞間的一類獨特的淋巴細胞群。IELs由多種亞群構成,主要包括TCRγδ+T細胞、TCRαβ+T細胞。TCRαβ+IELs又可分為TCRαβ+CD8aa+IELs、 TCRαβ+CD8αβ+IELs和TCRαβ+CD4+IELs三個亞群；TCRγδ+幾乎全部為CD8αα+。IELs的多亞群構成,也提示了它們來源的多樣性。因此,IELs來源多樣,功能復雜,已成為近年來腸道免疫研究的熱點。在生理情況下,IELs分泌細胞因子(如KGF)、抗菌肽等,維持上皮細胞生長及抵御病原體入侵；病理情況下,IELs快速做出應答,從半活化狀態(tài)迅速進入活化狀態(tài),分泌細胞因子參與炎癥反應,同時這些細胞因子也可能介導對腸黏膜的損傷,導致屏障功能下降。前期研究發(fā)現(xiàn),小腸I/R可以直接損傷腸上皮細胞以及細胞間連接。例如：腸黏膜絨毛的明顯斷裂、脫落和腸道上皮細胞的凋亡增加、壞死；維持IELs在上皮細胞間定植的緊密連接蛋白(Claudin-1和Occludin)結構破壞；腸黏膜通透性增加。我們在病人缺血的腸道標本中發(fā)現(xiàn),IL-7表達增多。IL-7作為腸上皮細胞分泌的、刺激淋巴細胞發(fā)育的細胞因子,在維持IELs表型和功能方面發(fā)揮了重要作用。在我們課題組前期構建的IL-7腸道過表達小鼠中發(fā)現(xiàn),IELs表現(xiàn)為Thl型細胞因子分泌增多,活化標志CD69+表達上調。提示：IL-7的上調,介導淋巴細胞過度活化,參與炎癥反應。IELs無論空間距離和功能調控都與腸上皮細胞有著緊密聯(lián)系。因此,我們推測I/R可導致IELs的過度活化,同時,活化后的IELs介導了對腸黏膜屏障的損傷。我們在本論文中重點探討小腸I/R條件下IELs的功能變化,以及其對上皮屏障產生的影響。同時,我們也注意到,急性放射性損傷可以導致上皮細胞大量死亡,由上皮細胞分泌的IL-7是否會受到影響。那么,這種情況下,IELs又將發(fā)生什么變化呢?我們針對這一問題也將進行初步的探討。因此本研究運用流式細胞分選,IELs上清與上皮細胞共培養(yǎng)、免疫組化、蛋白印跡、ELISA等技術,探討I/R和急性放射性損傷條件下(以I/R為重點),IELs亞型及功能的變化規(guī)律,以及IELs功能改變后對腸黏膜屏障損傷的相關機制。其中包括：明確對上皮屏障造成損傷的IELs源性細胞因子,該細胞因子對緊密連接蛋白如Claudin-1、Occludin等的表達及結構變化情況；并通過檢測跨上皮電阻抗(TER),確定IELs對屏障功能的調控作用；最后人為干預該細胞因子,評估腸屏障損傷的改善情況。為進一步闡明腸急性損傷條件下IELs的變化規(guī)律及其調控腸黏膜屏障的作用機制提供理論依據(jù),為腸黏膜屏障損傷干預提供新靶點。方法：建立小鼠急性小腸I/R模型及急性放射性損傷模型,運用HE染色,免疫組化,Ussing Chamer, Western Blot等技術了解急性損傷情況下腸黏膜的改變情況。圍繞腸I/R模型,運用流式細胞技術、免疫組化、定量PCR技術,闡明I/R條件下IELs表型和功能變化規(guī)律。運用流式細胞分選IELs,結合IELs上清與上皮細胞共培養(yǎng)模型,明確IELs源性細胞因子TNF-a對上皮細胞的作用,以及細胞因子對上皮間緊密連接蛋白表達及結構的影響。使用抗TNF-a抗體術前干預,通過跨上皮電阻抗(TER)及組織損傷評分的技術手段,明確IELs源細胞因子在腸黏膜屏障損傷中發(fā)揮了關鍵作用。結果：腸I/R條件下,小腸黏膜正常結構受損,絨毛斷裂,上皮下間隙增大,上皮脫落,固有層細胞成分增多,腸黏膜通透性增大。腸I/R后腸黏膜緊密連接蛋白(Claudin-1, Occludin)表達下降,Occludin在上皮細胞頂端正常結構遭到破壞。急性腸I/R對IELs數(shù)量沒有影響,但導致IELs與上皮細胞的正常連接消失。腸I/R條件下,上皮細胞分泌IL-7增多,IELs亞群CD8ap+, CD4+和TCRa(3+的百分比增高,CD8+, CD4+ IELs凋亡先后開始啟動。并且CD8+, CD4+ IELs活化顯著增加,TNF-α、IL-10mRNA拷貝數(shù)增多,KGF減少,IFN-y沒有明顯變化。I/R處理后的IELs上清與上皮細胞共培養(yǎng)后,發(fā)現(xiàn)上皮細胞Claudin-1、Occludin表達下降,Occludin結構完整性破壞,上皮屏障通透性增高,在I/R處理后的IELs上清中加入TNF-a抗體,可以逆轉上述改變。I/R處理后,CD45+IELs胞內合成以及分泌的TNF-a增多,用IL-7刺激體外培養(yǎng)的IELs后,TNF-a分泌明顯增多�？筎NF-a治療可以顯著緩解I/R小鼠腸黏膜損傷。急性放射性腸損傷直接導致腸黏膜結構破壞嚴重,絨毛形態(tài)不能正常維持,凋亡細胞增多,腸道IL-7表達降低,腸道IELs細胞數(shù)量72h后減少非常明顯。給予外源性IL-7處理,急性放射性腸損傷導致的IELs數(shù)量降低得到改善。結論：急性I/R條件下：IELs受到影響,表現(xiàn)為與上皮細胞間連接結構破壞,IELs亞群比例失調；IELs活化增加,其分泌的TNF-a介導了對上皮細胞的損傷以及上皮間緊密連接蛋白正常結構的破壞�？筎NF-a治療可以顯著改善腸黏膜損傷。急性放射性腸損傷條件下：腸黏膜結構嚴重破壞,絨毛形態(tài)不能維持,凋亡細胞增多,腸道上皮表達IL-7降低是IELs細胞數(shù)量減少的重要因素之一,給予外源性IL-7治療可以緩解IELs數(shù)量降低,改善放射損傷后腸黏膜免疫功能。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R574
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1 石辛甫,高杰英;腸上皮間淋巴細胞的研究進展[J];免疫學雜志;1998年04期
2 曾小川，，汪維偉;胚胎腸上皮的組織發(fā)生機制之研究進展[J];重慶醫(yī)科大學學報;1998年03期
3 Trier J S ,楊希山;腸M細胞的結構和功能[J];國外醫(yī)學(消化系疾病分冊);1992年03期
4 周靜,王杉,葉穎江,楊q
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