潘氏细胞在肠道稳态中的作用

肠道稳态对维持肠道正常生理功能具有重要意义。位于肠道上皮隐窝的潘氏细胞是肠道上皮屏障的重要组成部分,它们分泌大量生物效应分子,为小肠干细胞提供小生境以及调控肠道菌群。炎症性肠炎克罗恩病经常伴随潘氏细胞功能异常,Nod2、Xbp-1、Atg16L1、KCNN4等克罗恩病的易感基因在潘氏细胞中高表达,并调控潘氏细胞的重要生理活动。对潘氏细胞的研究有望揭示维持肠道稳态的生理机制,攻克炎症性肠炎等疾病的难关。     

营养对肠道干细胞增殖分化的调节作用

肠道上皮细胞功能的发挥依赖于定植在隐窝底部肠道干细胞(intestinal stem cells,ISCs)正常的增殖和分化。ISCs周边细胞及其相关生长因子共同组成了干细胞巢,调控ISCs的功能。来自于肠腔中的营养素也能够作用于肠道干细胞,调节肠道干细胞的增殖功能,从而影响肠道上皮的形态及功能。以Wnt/β-catenin等为代表的信号转导途径参与了上述调节过程。该文综述了近年来不同营养素对肠道干细胞增殖分化的影响,为实现肠道干细胞增殖分化的营养调控提供新的思路。     

信号分子通路网络对肠干细胞增殖和分化的调控

肠道具有营养吸收和不断更新的屏障保护双重优势,而肠道隐窝底部的干细胞是其实现多重生理功能的结构基础。本文总结当前对肠干细胞(intestinal stem cells,ISCs)的增殖分化影响的相关研究,罗列了Notch信号通路、BMP信号通路、Wnt信号通路、EGF信号通路及Hippo信号通路对ISCs增殖和分化的影响,其中Notch信号:维持ISCs并平衡分泌系祖细胞与吸收性祖细胞; BMP信号:调控ISCs分化并改变EEC细胞亚型分泌的激素谱系; Wnt信号:调控ISCs增殖; EGF信号:调控ISCs增殖速率; Hippo信号:调控ISCs增殖和分化,并且相关信号通路之间形成交叉互作网络,以协调ISCs增殖与分化,维持肠道的生理功能。     

隐窝干细胞和结肠癌的发生

肠道上皮细胞系是人体细胞更新最快的组织,更新速率甚至远远超过了肿瘤组织,这种无与伦比的更新速率如同一把双刃剑,一方面可以迅速的更新和修复肠粘膜,另一方面却大大增加了肠道细胞恶化的易感性。Wnt信号、Notch信号、BMP信号都参与了隐窝干细胞增殖分化的平衡,它们中任何一个组分发生突变或异常都将会导致结肠癌的发生。结肠癌的发生很可能是肠隐窝干细胞分化受阻的结果,隐窝干细胞是致瘤起始事件或突变的标靶。     

肠道干细胞及其干细胞巢

肠上皮是动物体内更新速度最快的组织之一,其功能的正常发挥有赖于定植在隐窝底部的肠道干细胞。肠道干细胞周围的细胞构成干细胞巢,为其提供了支持性微环境。随着肠道干细胞标记分子、谱系示踪技术、类器官培养等技术的进步和完善,使得人们对于肠道干细胞来源、增殖、分化相关信号通路的认识不断加深。该文就近年来肠道干细胞及其干细胞巢的研究进展进行了简要综述。     

肠道隐窝-绒毛轴上皮细胞更新及调控机制研究进展

肠道不仅是营养物质消化吸收的主要部位,也是重要的免疫器官和内分泌器官.小肠上皮细胞的分化对于肠道应激后的损伤修复、免疫屏障以及肠道功能的正常行使具有非常重要的意义.近年来,肠道上皮隐窝-绒毛轴干细胞自我更新、分化和调控的研究得到了快速发展.本文结合本研究组的研究成果综述了哺乳动物肠道隐窝-绒毛轴上皮细胞分化过程中差异基因和蛋白表达;信号通路、转录因子和表观遗传修饰对肠上皮细胞分化的影响以及营养因子对肠道细胞分化和损伤修复调控的最新研究进展,以期在营养学和药理学方面,为干预和治疗肠道损伤及相关疾病提供理论指导依据.     

肠道菌群：肠道干细胞增殖分化的调节者

肠道干细胞(intestinal stem cells, ISCs)是肠道各类上皮细胞的来源,通过平衡增殖与分化维持肠道稳态。同时,肠道菌群及其代谢物在维持宿主肠道稳态中也发挥着重要作用。随着技术的发展,研究者认识到ISCs与肠道菌群之间存在相互作用。研究表明,ISCs对上皮细胞亚型的调控影响肠道菌群的组成,并且肠道菌群及其代谢物也影响ISCs介导的上皮发育。本文阐述了ISCs分化对肠道菌群的影响,重点总结了肠道菌群及其代谢物调控ISCs增殖分化的研究进展,从菌群调控ISCs的角度探讨肠道损伤的治疗思路,并对未来可能的研究方向进行讨论。     

饲料精氨酸水平对斜带石斑鱼幼鱼生长和肠道形态的影响

正精氨酸作为一种鱼类生长发育所必需的碱性氨基酸,是机体合成蛋白质的重要成分[1],对肠细胞的增殖分化和维持细胞形态结构具有重要的保护作用[2]。对陆生动物如羊[2]、仔猪[3,4]和鼠[5]等的研究表明,日粮中添加精氨酸能显著提高肠道绒毛高度和隐窝深度,从而提高肠道消化吸收能力。目前,在鱼类的相关研究中,Cheng等[6]报道在饲料中添加精氨酸或谷氨酰胺能显著提高杂交条纹鲈(Motone chrysopsxMorone,saxatilis)和美国红鱼(Sciaenops ocellatus)[7]前肠和中肠的皱襞和微绒毛高度,增大肠道吸收面积。肠道是动物吸收利用精氨酸的     

ToxoDB#17型弓形虫感染小鼠引起小肠潘氏细胞溶菌酶缺失

为探究Toxo DB#17型弓形虫对昆明小鼠小肠潘氏细胞(Paneth cells,PCs)溶菌酶的表达及小肠病理损伤特点,该研究以8周龄小鼠为研究对象,灌胃1×106个Toxo DB#17型弓形虫卵囊,分别在灌胃后6 HAI(hours after inoculation)、1 DAI(day after inoculation)、3 DAI、8 DAI取小肠各段,常规方法制作石蜡切片,HE和免疫组化染色,研究小肠病理损伤、虫体分布、潘氏细胞及溶菌酶表达特点。结果显示,小鼠弓形虫感染率为100%,弓形虫虫体抗原分布随时间延长呈增多趋势(P0.05),小肠的病理损伤随着弓形虫感染时间的延长,未见明显变化。小肠隐窝数、含潘氏细胞隐窝数、PCs总数和颗粒总数的变化总趋势呈现先减少后增加再减少的趋势,3 DAI数量较多(P0.05),潘氏细胞颗粒未观察到溶菌酶阳性染色反应。以上结果表明,Toxo DB#17型弓形虫抑制潘氏细胞溶菌酶的表达,对肠道损伤较轻,潘氏细胞及其分泌颗粒对弓形虫有应答反应,溶菌酶的缺失与Toxo DB#17型弓形虫成功入侵肠道有关。     

肠道类器官在精准医学中的应用

类器官3D培养是一种新兴的用来研究组织成体干细胞生长、分化、器官形成的体外研究系统.目前,肠道类器官的3D培养是将分离的肠道隐窝或干细胞植入含有多种生长因子的基质胶中,在基质3D支撑下生成具有肠道上皮样结构的微型空心球体,这些球体被称为肠道类器官.该类器官包含有所有种类的肠道功能上皮细胞,能最大程度模拟肠道组织,故也称之为"迷你肠".结直肠肿瘤细胞也可以利用该3D体系培养得到肿瘤类器官.这些肠道类器官可被广泛应用于炎症性肠病、肠道损伤再生、肠癌等多种肠道疾病的研究.本综述讨论了关于肠道干细胞的最新研究进展,正常类器官和肿瘤类器官的培养,同时还将探讨类器官在疾病建模和组织再生、基因修复、肿瘤个性化治疗等精准医学方面的应用.     

潘氏细胞研究进展

潘氏细胞是位于小肠腺底部的浆液性腺上皮细胞,其主要特征是细胞顶部有大量粗大的嗜酸性分泌颗粒,内含防御素、溶菌酶、sIgA等多种抗菌物质。表达于潘氏细胞的NOD2、Toll样受体9、肝癌-肠-胰腺／胰腺炎相关蛋白、RegⅢγ、肿瘤坏死因子仅、粒细胞-巨噬细胞集落刺激因子、白介素-17等也是免疫与炎症反应的重要成分。金属硫蛋白、富半胱氨酸肠蛋白、潘氏细胞锌结合蛋白等金属结合蛋白均分布于潘氏细胞,提示潘氏细胞参与金属代谢。潘氏细胞是构成肠黏膜屏障的重要细胞成分。NOD2单核苷酸多态性与克罗恩病有关。潘氏细胞化生常发生于胃、大肠的炎症与肿瘤病变,其病理意义有待于进一步研究。     

Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth.     

Proteomic Profiling of Enteroid Cultures Skewed toward Development of Specific Epithelial Lineages

Recently, 3D small intestinal organoids (enteroids) have been developed from cultures of intestinal stem cells which differentiate in vitro to generate all the differentiated epithelial cell types associated with the intestine and mimic the structural properties of the intestine observed in vivo. Small‐molecule drug treatment can skew organoid epithelial cell differentiation toward particular lineages, and these skewed enteroids may provide useful tools to study specific epithelial cell populations, such as goblet and Paneth cells. However, the extent to which differentiated epithelial cell populations in these skewed enteroids represent their in vivo counterparts is not fully understood. This study utilises label‐free quantitative proteomics to determine whether skewing murine enteroid cultures toward the goblet or Paneth cell lineages results in changes in abundance of proteins associated with these cell lineages in vivo. Here, proteomics data confirms that skewed enteroids recapitulate important features of the in vivo gut environment, demonstrating that they can serve as useful models for the investigation of normal and disease processes in the intestine. Furthermore, comparison of mass spectrometry data with histology data contained within the Human Protein Atlas identifies putative novel markers for goblet and Paneth cells.     

Thyroid Hormone Regulation of Adult Intestinal Stem Cell Development: Mechanisms and Evolutionary Conservations

The adult mammalian intestine has long been used as a model to study adult stem cell function and tissue renewal as the intestinal epithelium is constantly undergoing self-renewal throughout adult life. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells located in the crypt. The development of this self-renewal system is, however, poorly understood. A number of studies suggest that the formation/maturation of the adult intestine is conserved in vertebrates and depends on endogenous thyroid hormone (T3). In amphibians such as Xenopus laevis, the process takes place during metamorphosis, which is totally dependent upon T3 and resembles postembryonic development in mammals when T3 levels are also high. During metamorphosis, the larval epithelial cells in the tadpole intestine undergo apoptosis and concurrently, adult epithelial stem/progenitor cells are formed de novo, which subsequently lead to the formation of a trough-crest axis of the epithelial fold in the frog, resembling the crypt-villus axis in the adult mammalian intestine. Here we will review some recent molecular and genetic studies that support the conservation of the development of the adult intestinal stem cells in vertebrates. We will discuss the mechanisms by which T3 regulates this process via its nuclear receptors.     

Gut commensal bacteria,Paneth cells and their relations to radiation enteropathy

In steady state, the intestinal epithelium forms an important part of the gut barrier to defend against luminal bacterial attack. However, the intestinal epithelium is compromised by ionizing irradiation due to its inherent self-renewing capacity. In this process, small intestinal bacterial overgrowth is a critical event that reciprocally alters the immune milieu. In other words, intestinal bacterial dysbiosis induces inflammation in response to intestinal injuries, thus influencing the repair process of irradiated lesions. In fact, it is accepted that commensal bacteria can generally enhance the host radiation sensitivity. To address the determination of radiation sensitivity, we hypothesize that Paneth cells press a critical “button” because these cells are central to intestinal health and disease by using their peptides, which are responsible for controlling stem cell development in the small intestine and luminal bacterial diversity. Herein, the most important question is whether Paneth cells alter their secretion profiles in the situation of ionizing irradiation. On this basis, the tolerance of Paneth cells to ionizing radiation and related mechanisms by which radiation affects Paneth cell survival and death will be discussed in this review. We hope that the relevant results will be helpful in developing new approaches against radiation enteropathy.     

Genetic Ablation of Afadin Causes Mislocalization and Deformation of Paneth Cells in the Mouse Small Intestinal Epithelium

Afadin is an actin filament-binding protein that acts cooperatively in cell adhesion with the cell adhesion molecule nectin, and in directional cell movement with the small G protein Rap1 in a nectin-independent manner. We studied the role of afadin in the organization of the small intestinal epithelium using afadin conditional gene knockout (cKO) mice. Afadin was localized at adherens junctions of all types of epithelial cells throughout the crypt-villus axis. Paneth cells were localized at the base of the crypt in control mice, but not confined there, and migrated into the villi in afadin-cKO mice. The distribution of other types of epithelial cells did not change significantly in the mutant mice. The Paneth cells remaining in the crypt exhibited abnormal shapes, were buried between adjacent cells, and did not face the lumen. In these cells, the formation of adherens junctions and tight junctions was impaired. Rap1 and EphB3 were highly expressed in control Paneth cells but markedly down-regulated in the afadin-deficient Paneth cells. Taken together, the results indicate that afadin plays a role in the restricted localization of Paneth cells at the base of the crypt by maintaining their adhesion to adjacent crypt cells and inhibiting their movement toward the top of villi.     

Assessment of the symmetry of stem-cell mitoses.

A model of Paneth-cell renewal in the small intestinal epithelium is used to estimate the probability that epithelial stem-cell mitoses are symmetric in the sense that they produce two cells of the same type. I found that counts of the number of Paneth cells per crypt (Paneth cells are terminally differentiated cells derived from small intestinal epithelial stem cells) support a model in which most, if not all, stem-cell mitoses are symmetric.     

Intrauterine Growth Restriction Alters Mouse Intestinal Architecture during Development

Infants with intrauterine growth restriction (IUGR) are at increased risk for neonatal and lifelong morbidities affecting multiple organ systems including the intestinal tract. The underlying mechanisms for the risk to the intestine remain poorly understood. In this study, we tested the hypothesis that IUGR affects the development of goblet and Paneth cell lineages, thus compromising the innate immunity and barrier functions of the epithelium. Using a mouse model of maternal thromboxane A₂-analog infusion to elicit maternal hypertension and resultant IUGR, we tested whether IUGR alters ileal maturation and specifically disrupts mucus-producing goblet and antimicrobial-secreting Paneth cell development. We measured body weights, ileal weights and ileal lengths from birth to postnatal day (P) 56. We also determined the abundance of goblet and Paneth cells and their mRNA products, localization of cellular tight junctions, cell proliferation, and apoptosis to interrogate cellular homeostasis. Comparison of the murine findings with human IUGR ileum allowed us to verify observed changes in the mouse were relevant to clinical IUGR. At P14 IUGR mice had decreased ileal lengths, fewer goblet and Paneth cells, reductions in Paneth cell specific mRNAs, and decreased cell proliferation. These findings positively correlated with severity of IUGR. Furthermore, the decrease in murine Paneth cells was also seen in human IUGR ileum. IUGR disrupts the normal trajectory of ileal development, particularly affecting the composition and secretory products of the epithelial surface of the intestine. We speculate that this abnormal intestinal development may constitute an inherent “first hit”, rendering IUGR intestine susceptible to further injury, infection, or inflammation.     

Cloning of intestinal phospholipase A2 from intestinal epithelial RNA by differential display PCR

Differential display polymerase chain reaction (DD-PCR) is a powerful technique for comparing gene expression between cell types, or between stages of development or differentiation. Differentially expressed genes may be cloned and analysed further. Here we extend the use of DD-PCR to analyse differences in gene expression between two complex epithelia: that of the small intestine and of the large intestine. The aim of this study was to identify genes expressed preferentially in Paneth cells. Paneth cells are secretory epithelial cells putatively involved in host defense and regulation of crypt cell proliferation and are found at the base of the small intestinal crypts adjacent to the stem cell zone. Of 34 clones that were analysed, partial sequencing identified two clones related to known Paneth cell products: a homologue of secretory phospholipase A2 (clone B1) and a homologue of a neutrophil defensin (clone C5). B1 was strongly expressed in Paneth cells, as demonstrated by in-situ hybridization. B1 was also expressed at a lower level in the large intestinal epithelium. A full length B1 cDNA clone was isolated and sequenced, and shown to be highly homologous to type II secretory phospholipase A2 genes, and almost identical to the enhancing factor gene and the putative gene for the MOM-1 locus. B1 expression is limited to the intestinal tract, and we propose that it be designated intestinal phospholipase A2, or i -PLA2. The method we describe is well suited to the rapid identification of genes expressed exclusively or predominantly in Paneth cells.