肠上皮内淋巴细胞分离鉴定

肠上皮内淋巴细胞（intestine intraepithelial lymphocytes，iIEL），是机体内数量最多的淋巴细胞群体之一。在黏膜免疫防御和口服免疫耐受形成等方面发挥重要的作用。本文参考国内外多种方法，选用机械和消化法分离细胞、密度梯度离心法纯化iIEL，摸索快速、高效、稳定的iIEL提取方法，并对其增殖和表型进行了初步分析。     

沉默信息调节因子6基因敲除模型小鼠肠黏膜形态及转录组学的特征性变化

背景:肠黏膜更新是维持机体稳态的关键生理过程,其中隐窝干细胞是肠上皮增殖和分化的驱动力.沉默信息调节因子(silent information regulator,SIRT)参与细胞的基因修复、代谢、能量平衡和寿命的调节,在肠上皮稳态中的研究逐步成为热点.肠类器官由单个隐窝干细胞经3D培养形成,可体外呈现肠上皮更新及受...     

细胞焦亡在肠黏膜屏障损伤机制中的作用

肠上皮细胞、免疫系统及微生物的相互联系维持肠黏膜屏障完整性和自我更新。肠黏膜屏障损伤易导致难以预测的严重疾病发生。细胞焦亡是一种由活化的半胱天冬酶切割Gasdermin家族蛋白介导的炎症细胞死亡形式,在急慢性疾病控制中发挥重要作用。病理生理状态下,细胞焦亡参与肠黏膜屏障损伤及异常免疫调节,最终导致多种肠道疾病发生。本文综述细胞焦亡在肠道感染、肠缺血再灌注、坏死性小肠结肠炎及炎症性肠病中的最新调控机制,针对此类肠道疾病提供新的有效治疗策略。     

黏蛋白-2与肠黏膜屏障损伤的研究进展

黏蛋白-2(MUC2)是肠黏液层主要成分,覆盖于肠上皮细胞顶端,主要由杯状细胞分泌,在润滑肠道、为肠内抗菌蛋白及共生菌群提供黏附位点、抵御肠内致病菌及有害物质入侵等方面发挥着重要功能.近年来MUC2在肠道黏膜屏障损伤中的作用研究日趋受到学者重视,这也可能是肠黏膜屏障损伤的一个重要治疗突破口.     

肠上皮内淋巴细胞及其与肠道疾病

肠上皮内淋巴细胞 (iIEL)是一组庞大的T细胞群体 ,它的表型、功能及其发育均与经胸腺发育成熟的T细胞不同 ,iIEL在有病毒感染、体外刺激和寄生虫感染引起的肠道疾病中起重要的免疫防御和免疫调节作用     

细胞因子与肠上皮内淋巴细胞的发育分化和生物学功能

肠上皮内淋巴细胞由多种T淋巴细胞亚群构成 ,部分亚群被认为是在胸腺外发育分化的。IL 7与IL 7R之间的信号传递对TCRγδ iIEL的发育分化是必不可少的。IL 15也可能对iIEL在胸腺外的发育和维持起重要的调节作用。iIEL能分泌多种细胞因子 ,在抗感染免疫和调节肠上皮细胞功能方面起重要作用。     

一种新型肠上皮干细胞分离方法的建立

目的 探讨密度梯度离心法作为一种新型肠上皮干细胞分离方法的可行性.方法 通过腹腔注射大剂量5-氟尿嘧啶(5-FU)制备肠黏膜严重损伤小鼠模型,取出损伤的肠黏膜消化成单细胞悬液,利用Percoll密度梯度离心法分离细胞体积大小不同的细胞群.HE染色观察各群细胞形态特征,免疫细胞化学及RT-PCR检测各群细胞musashi-1(msi-1)的表达.结果 分离的大部分细胞位于50%和70%密度梯度的Percoll分离液体层,50%密度梯度分离液体层中的细胞符合干细胞的形态特征,免疫细胞化学检测其msi-1表达阳性率约为93%,RT-PCR显示该群细胞msi-1 mRNA表达较强.结论 建立了一种简便、有效的分离具有活性的肠上皮干细胞的方法.     

自噬在肠黏膜屏障功能作用机制的研究进展

自噬是细胞通过膜囊泡结构降解胞质内大分子物质和受损细胞器维持机体稳态的生物学过程。在肠黏膜屏障功能发生障碍过程中,自噬对于维持肠上皮细胞的存活起关键性作用。负调控自噬可导致肠道炎性反应和肿瘤的发生。     

缺血再灌注条件下BMP2/4参与肠黏膜屏障损伤的研究

目的探讨缺血再灌注条件下BMP2/4参与肠黏膜屏障损伤的机制。方法制作肠上皮细胞缺氧模型、大鼠肠缺血再灌注损伤模型。取空肠切片,Western blot、免疫荧光检测肠上皮细胞内BMP的变化; Western blot、免疫荧光、RT-PCR检测外源性BMP蛋白对肠上皮细胞内NF-κB信号及TNF-a、IL-6的表达变化的影响; Western blot检测外源性BMP蛋白对紧密连接蛋白occludin表达变化的影响。结果缺氧及缺血再灌注条件下肠上皮细胞内的BMP2/4表达明显增加,BMP2/4通过激活肠上皮细胞内的NF-κB信号导致炎症介质TNF-a、IL-6表达增加,增加肠黏膜屏障的通透性及减少紧密连接蛋白occludin的表达,损害肠黏膜物理屏障的完整性,从而造成肠黏膜屏障的损伤。结论缺血再灌注条件下,BMP信号通过激活NF-κB参与肠黏膜屏障损伤。     

053 肠上皮内淋巴细胞及其与肠道疾病

肠上皮内淋巴细胞(iIEL)是一组庞大的T细胞群体,它的表型、功能及其发育均与经胸腺发育成熟的T细胞不同,iIEL在有病毒感染、体外刺激和寄生虫感染引起的肠道疾病中起重要的免疫防御和免疫调节作用.     

Caspases-3, -8, and -9 are required for induction of epithelial cell apoptosis by enteropathogenic E. coli but are dispensable for increased paracellular permeability

Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhea, particularly among infants in developing countries. An increase in intestinal permeability due to EPEC infection has been suggested as a factor in the development of diarrhea. Abnormally high levels of programmed cell death (apoptosis) of intestinal epithelial cells can lead to increased intestinal permeability. The effects of EPEC on cell apoptosis remain incompletely understood. This study characterized the mechanisms of EPEC-induced epithelial apoptosis and examined whether this effect contributes to heightened permeability in an in vitro model of infection. We report that EPEC-induced apoptosis in T84 intestinal epithelial cells via a mechanism involving caspases-3, -6, -8, and -9, the cleavage of PARP, and oligonucleosome formation. In addition, EPEC time-dependently increased paracellular permeability as assessed by transepithelial resistance and the apical-to-basolateral movement of 3000 MW dextran. Furthermore, EPEC infection led to the cleavage and mislocalization of tight junctional ZO-1 and occludin. However, pharmacological inhibition of caspases did not prevent the EPEC-induced disruptions in epithelial barrier structure and function. Taken together, these results suggest that a caspase-dependent upregulation in epithelial cell apoptosis during EPEC infection occurs independent of impaired intestinal barrier function.     

The role of innate signaling in the homeostasis of tolerance and immunity in the intestine

In the intestine innate recognition of microbes is achieved through pattern recognition receptor (PRR) families expressed in immune cells and different cell lineages of the intestinal epithelium. Toll-like receptor (TLR) and nucleotide-binding and oligomerization domain-like receptor (NLR) families are emerging as key mediators of immunity through their role as maturation factors of immune cells and triggers for the production of cytokines and chemokines and antimicrobial factors. At the mucosal surface chronic activation of the immune system is avoided through the epithelial production of a glycocalyx, steady-state production of antimicrobial factors as well as the selective expression and localization of PRRs. Additionally, the polarization of epithelial TLR signaling and suppression of NF-κB activation by luminal commensals appears to contribute to the homeostasis of tolerance and immunity. Several studies have demonstrated that TLR signaling in epithelial cells contributes to a range of homeostatic mechanisms including proliferation, wound healing, epithelial integrity, and regulation of mucosal immune functions. The intestinal epithelium appears to have uniquely evolved to maintain mucosal tolerance and immunity, and future efforts to further understand the molecular mechanisms of intestinal homeostasis may have a major impact on human health.     

Oral tolerance to haptens: intestinal epithelial cells from 2,4-dinitrochlorobenzene-fed mice inhibit hapten-specific T cell activation in vitro

The mechanisms underlying the induction of immunological tolerance after feeding soluble exogenous antigens, including proteins and haptens, are still unclear. Using a model of oral tolerance to the contact-sensitizing hapten 2,4-dinitrochlorobenzene (DNCB), we have compared the ability of intestinal epithelial cells and of Peyer's patch APC to present DNCB in vitro or ex vivo after oral feeding, to specific peripheral lymph node T cells from DNCB-sensitized mice. In contrast to Peyer's patch APC, which induce efficient hapten-specific T cell activation upon exposure to the hapten either in vitro or in vivo, mature MHC class-II-positive intestinal epithelial cells were unable to induce T cell activation in either case. Interestingly, enterocytes from DNCB-fed mice exerted a dramatic inhibitory effect on the proliferative response of hapten-primed T cells in response to dinitrobenzene sulfonate presented by syngeneic spleen cells. This inhibitory effect, which was also observed with supernatant of intestinal epithelial cells from DNCB-fed mice, could be reversed by neutralizing anti-transforming growth factor (TGF)-β antibodies. In addition, pre-incubation of hapten-sensitized T cells with enterocytes from DNCB-fed mice induced T cell anergy, which could be reversed by exogenous interleukin-2 or interleukin-4. These data demonstrate that intestinal epithelial cells activated in vivo by oral administration of DNCB are able to block proliferation of activated T cells through secretion of immunosuppressive cytokines such as TGF-β. It is proposed that intestinal epithelial cells may play a significant role in oral tolerance by limiting T cell-mediated hypersensitivity responses.     

T cells,dendritic cells and epithelial cells in intestinal homeostasis

The mucosal immune system of the intestinal tract is continuously exposed to both potential pathogens and beneficial commensal microorganism. A variety of mechanisms contribute to the ability of the gut to either react or remain tolerant to antigen present in the intestinal lumen. Antigens of the gut commensals are not simply ignored, but rather trigger an active immunosuppressive process, which prevents the outcome of immunopathology. The aim of this review is to provide an update on the mechanism of intestinal homeostasis, with particular focus on the complex crosstalk between T cells, dendritic cells and intestinal epithelial cells.     

Mucosal innate immune cells regulate both gut homeostasis and intestinal inflammation

Continuous exposure of intestinal mucosal surfaces to diverse microorganisms and their metabolites reflects the biological necessity for a multifaceted, integrated epithelial and immune cell‐mediated regulatory system. The development and function of the host cells responsible for the barrier function of the intestinal surface (e.g., M cells, Paneth cells, goblet cells, and columnar epithelial cells) are strictly regulated through both positive and negative stimulation by the luminal microbiota. Stimulation by damage‐associated molecular patterns and commensal bacteria‐derived microbe‐associated molecular patterns provokes the assembly of inflammasomes, which are involved in maintaining the integrity of the intestinal epithelium. Mucosal immune cells located beneath the epithelium play critical roles in regulating both the mucosal barrier and the relative composition of the luminal microbiota. Innate lymphoid cells and mast cells, in particular, orchestrate the mucosal regulatory system to create a mutually beneficial environment for both the host and the microbiota. Disruption of mucosal homeostasis causes intestinal inflammation such as that seen in inflammatory bowel disease. Here, we review the recent research on the biological interplay among the luminal microbiota, epithelial cells, and mucosal innate immune cells in both healthy and pathological conditions.     

Intestinal epithelial barrier functions in ageing

The intestinal epithelial barrier protects the mucosa of the gastrointestinal (GI)-tract and plays a key role in maintaining the host homeostasis. It encompasses several elements that include the intestinal epithelium and biochemical and immunological products, such as the mucus layer, antimicrobial peptides (AMPs) and secretory immunologlobulin A (sIgA). These components are interlinked with the large microbial community inhabiting the gut to form a highly sophisticated biological system that plays an important role on many aspects of human health both locally and systemically. Like any other organ and tissue, the intestinal epithelial barrier is affected by the ageing process. New insights have surfaced showing that critical functions, including intestinal stem cell regeneration and regulation of the intestinal crypt homeostasis, barrier integrity, production of regulatory cytokines, and epithelial innate immunity to pathogenic antigens change across life. Here we review the age-associated changes of the various components of the intestinal epithelial barrier and we highlight the necessity to elucidate further the mechanisms underlying these changes. Expanding our knowledge in this area is a goal of high medical relevance and it will help to define intervention strategies to ameliorate the quality of life of the ever-expanding elderly population.     

Intestinalization of gastric signet ring cell carcinomas with progression

 Recent developments in mucin histochemistry and immunohistochemistry have made reliable determination of the gastric and intestinal phenotypes of gastric carcinoma cells possible. Phenotypic expression changes from gastric epithelial cell type to intestinal epithelial cell type with the growth of gastric tumours in experimental animals. We studied cell differentiation in gastric signet ring cell carcinomas with progression in 203 surgically obtained specimens. The results showed that the proportion of gastric phenotype carcinomas, in which over 90% of the tissue consists of gastric epithelial cell type cells, decreases with the depth of invasion. The proportion of mixed phenotype carcinomas (between 10% and 90% of the tissue made up of gastric and/or intestinal epithelial cell type cells) increases. The intestinal phenotype (over 90% intestinal epithelial cell type carcinoma cells) was found in four carcinomas (about 2%) involving the serosa. No clear relationship was evident between phenotypic expression of carcinoma cells and the degree of intestinal metaplasia of the surrounding mucosa. Progression of gastric signet ring cell carcinomas is associated with a phenotypic shift from gastric to intestinal type expression. Received: 7 January 1997 / Accepted: 12 March 1997     

Undifferentiated columnar cells on the gastric interfoveolar crest: a previously undescribed observation

We have observed and defined morphometrically and histochemically an entity composed of groups of undifferentiated columnar cells on the interfoveolar crest of gastric mucosa. These cells are clearly distinct from either normal foveolar cells or regenerative epithelial cells associated with ulcer healing. They show a close association with atrophic gastritis, particularly in the presence of type 3 sulphomucin-secreting intestinal metaplasia. We have termed this the gastric tip lesion since it is observed only on the tips of the mucosal folds. This has not been described previously. We propose that these cells are the precursors of type 3 intestinal metaplasia and may also provide a link between this type of intestinal metaplasia and the intestinal variant of gastric adenocarcinoma.     

IL-15 protects intestinal epithelial cells

IL-15, a T-cell growth factor, has been shown to be increased in inflammatory bowel disease (IBD). It has been suggested that neutralization of IL-15 could protect from T cell-dependent autoimmune inflammation. On the other hand, an anti-apoptotic effect of IL-15 has been demonstrated in kidney epithelial cells during nephritis. We therefore tested the role of IL-15 in two different experimental models of colitis in vivo, and in models of intestinal epithelial cell (IEC) apoptosis in vitro. IL-15 blockade in chronic dextran sulphate sodium-induced colitis resulted in aggravation of the disease with a significantly 2.1-fold increased epithelial damage score compared to controls. TUNEL staining clearly revealed increased apoptosis. IL-6, TNF and IFN-gamma secretion by mesenteric lymph node cells were increased. In the T cell-dependent SCID transfer model of colitis IL-15 neutralization reduced the inflammatory infiltration and proinflammatory cytokine production. Despite that, the intestinal epithelial damage was not reduced. In vitro, IL-15 pre-incubation prevented up to 75% of CH11 antibody-induced apoptosis in SW-480 cells and reduced caspase-3 activity. According to this, endogenously produced IL-15 in chronic colitis does not only act as a proinflammatory cytokine but has at the same time the potential to reduce mucosal damage by preventing IEC apoptosis.