肠上皮干细胞研究进展

肠上皮是人体更新最快的组织,位于其底部隐窝结构中的干细胞是它快速更新的驱动力所在.Lgr5+细胞是最主要的肠上皮干细胞类群,它可以分化产生所有的肠上皮细胞类群.Lgr5+肠干细胞的命运受Wnt,BMP,Notch,EGF和Hippo等生长发育信号通路的调控,以及肠道微生物和人体代谢水平的影响.肠上皮干细胞的稳态维持与肠道疾病发生紧密相关,其功能缺失会引起消化吸收功能下降、炎症发生,而其过度激活导致的增生又会诱发肿瘤,形成结直肠癌.对肠上皮干细胞的研究既可以加深对于肠上皮快速更新机制的了解,又可以为治疗结直肠癌等肠道疾病提供新的启示.     

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。     

在体分离小鼠小肠隐窝、绒毛上皮细胞的生化完整性研究

目的:检测低温条件下用螯合剂沉淀法分离的小鼠小肠上皮隐窝和绒毛细胞是否具有生化完整性.方法:使用螯合剂在低温(冰浴)条件下分离和富集小肠上皮绒毛和隐窝细胞;抽提DNA、RNA和总蛋白,用电泳的方法检测完整性;用Real-time PCR检测溶菌酶Lysozyme的表达以判断隐窝、绒毛细胞富集程度.结果:低温条件下分离的肠上皮隐窝、绒毛细胞形态完整;基因组DNA完整,未出现明显的DNA ladder现象;富集细胞的RNA完整;富集隐窝、绒毛细胞的蛋白未降解,两组总蛋白具有表达谱差异性;隐窝细胞富集物溶菌酶mRNA表达水平较绒毛细胞富集物高30倍以上.结论:小肠隐窝绒毛的生物学性状可在低温螯合剂沉底法分离过程中得到保存,提示此方法可以用来分析生理和创伤痛理条件下小肠上皮基因和蛋白表达改变.     

哺乳动物肠上皮屏障中非编码RNAs的调控作用

哺乳动物肠上皮是一种拥有快速自我更新能力的组织,在维持机体免疫稳态与肠道应激后的损伤修复中发挥重要作用。源于隐窝底部的多能肠干细胞不断进行增殖、迁移与分化,并沿隐窝 绒毛轴向上移动,从而维持肠上皮完整性。该过程受严格而复杂的基因调控网络参与。越来越多的数据表明,肠上皮完整性受到广泛的非编码RNA的调控,主要包括肠黏膜再生、保护与上皮屏障功能等方面。本文重点讨论了两类非编码RNA(包括microRNAs和lncRNAs)转录后调控肠上皮屏障功能的研究进展。其中,miR-503、miR-146和lnc-uc.173、lnc-SPRY4-IT1、lnc-plncRNA1、lnc-Gata6等,能够促进肠黏膜的更新,增强上皮屏障功能;相反,miR-222、miR-29b、miR-195和lnc-H19与lnc-BC012900等,抑制肠上皮再生并破坏肠上皮屏障功能。miRNAs、mRNAs与lncRNAs间构成复杂的分子网络,共同调控肠上皮稳态。深入研究与肠上皮相关的miRNAs和IncRNAs分子及其作用机制,探寻引起肠黏膜炎症的关键分子靶标,为肠道炎症临床诊治提供新方向与新方法。     

哺乳动物肠上皮屏障中非编码RNAs的调控作用

哺乳动物肠上皮是一种拥有快速自我更新能力的组织,在维持机体免疫稳态与肠道应激后的损伤修复中发挥重要作用。源于隐窝底部的多能肠干细胞不断进行增殖、迁移与分化,并沿隐窝 绒毛轴向上移动,从而维持肠上皮完整性。该过程受严格而复杂的基因调控网络参与。越来越多的数据表明,肠上皮完整性受到广泛的非编码RNA的调控,主要包括肠黏膜再生、保护与上皮屏障功能等方面。本文重点讨论了两类非编码RNA(包括microRNAs和lncRNAs)转录后调控肠上皮屏障功能的研究进展。其中,miR-503、miR-146和lnc-uc.173、lnc-SPRY4-IT1、lnc-plncRNA1、lnc-Gata6等,能够促进肠黏膜的更新,增强上皮屏障功能;相反,miR-222、miR-29b、miR-195和lnc-H19与lnc-BC012900等,抑制肠上皮再生并破坏肠上皮屏障功能。miRNAs、mRNAs与lncRNAs间构成复杂的分子网络,共同调控肠上皮稳态。深入研究与肠上皮相关的miRNAs和IncRNAs分子及其作用机制,探寻引起肠黏膜炎症的关键分子靶标,为肠道炎症临床诊治提供新方向与新方法。     

小鼠小肠类器官体外培养体系的建立与应用

目的建立一种研究肠上皮细胞的体外模型—小肠类器官培养体系,探索其相关病理检测技术方法,为肠道相关疾病的体外研究提供便利平台。方法将小鼠肠上皮隐窝分离并培养成小肠类器官,体外模拟肠上皮的生长发育过程。通过制作石蜡切片,探索应用免疫组化技术以及基于基质胶中类器官三维水平免疫荧光技术对相应的增殖与分化信号进行检测。结果探索并建立了小肠类器官体外培养体系,应用石蜡切片免疫组化技术与三维水平免疫荧光技术能够准确检测小肠上皮结构的生长发育状态。结论小肠类器官体外培养体系的建立与免疫检测技术的应用,将逐渐使其成为人们研究肠道相关疾病最为有利的技术手段。     

机械力及力学信号转导影响干细胞命运的研究进展

干细胞作为一种未分化的祖细胞,目前已被广泛应用于开展组织损伤修复、再生以及干细胞特异谱系分化的研究.大量研究表明,干细胞所处的微环境对调控干细胞的生长和分化具有重要作用,多种溶液介质、细胞外基质和信号通路等参与了干细胞命运的调控.尽管已有大量研究证明,溶液介质(如激素和生长因子)在干细胞的生长和分化中发挥重要作用,但近年来越来越多的研究表明,机械力及力学信号转导同样在干细胞自我更新、分化、衰老和凋亡等细胞生理过程中起到重要的作用.本文将对机械应力响应的细胞基础、生物力学及力学信号调控干细胞自我更新和分化,以及生物力学调控干细胞命运可能的作用机制几个方面加以综述.     

小鼠肠腺剂量存活曲线的一种测定方法——单位面积肠腺计数

小肠上皮是一个细胞更新系统,它的干细胞在肠腺部位。经电离辐射作用后,肠腺内的干细胞受到破坏,引起小肠上皮细胞更新系统动态平衡的紊乱。经超致死剂量照射后小肠上皮脱落,绒毛裸露,引起典型的肠型放射病,小鼠于3-5天死亡(Quastler,H.1956年;Bond,V.P.1965)。因此,肠腺的辐射敏感性是一个值得研究的问题。本实验提出受照射小鼠肠腺的剂量存活曲线的一种测定方法。     

陷窝蛋白-1与干细胞衰老、自我更新的研究进展

近年来有关干细胞临床应用的研究十分广泛,干细胞移植治疗各类疾病也已取得可观的效果。但随着干细胞技术的快速发展,干细胞体外增殖导致的衰老也成为制约干细胞应用的一大难题。陷窝蛋白-1是细胞膜上的一种整合膜蛋白,是形成陷窝的主要膜内在蛋白。已有研究表明,陷窝蛋白-1在细胞衰老及自我更新调节中起重要的调控作用。本文简要综述了干细胞的自我更新和衰老机制及陷窝蛋白-1的促增殖及抗衰老作用,旨在为干细胞临床应用及干细胞组织工程研究提供理论依据。     

Regulation of intestinal stem cell fate specification

The remarkable ability of rapid self-renewal makes the intestinal epithelium an ideal model for the study of adult stem cells. The intestinal epithelium is organized into villus and crypt, and a group of intestinal stem cells located at the base of crypt are responsible for this constant self-renewal throughout the life. Identification of the intestinal stem cell marker Lgr5, isolation and in vitro culture of Lgr5+ intestinal stem cells and the use of transgenic mouse models have significantly facilitated the studies of intestinal stem cell homeostasis and differentiation, therefore greatly expanding our knowledge of the regulatory mechanisms underlying the intestinal stem cell fate determination. In this review, we summarize the current understanding of how signals of Wnt, BMP, Notch and EGF in the stem cell niche modulate the intestinal stem cell fate.     

Modulation of stem cell fate in intestinal homeostasis,injury and repair

The mammalian intestinal epithelium constitutes the largest barrier against the external environment and makes flexible responses to various types of stimuli. Epithelial cells are fast-renewed to counteract constant damage and disrupted barrier function to maintain their integrity. The homeostatic repair and regeneration of the intestinal epithelium are governed by the Lgr5⁺ intestinal stem cells (ISCs) located at the base of crypts, which fuel rapid renewal and give rise to the different epithelial cell types. Protracted biological and physicochemical stress may challenge epithelial integrity and the function of ISCs. The field of ISCs is thus of interest for complete mucosal healing, given its relevance to diseases of intestinal injury and inflammation such as inflammatory bowel diseases. Here, we review the current understanding of the signals and mechanisms that control homeostasis and regeneration of the intestinal epithelium. We focus on recent insights into the intrinsic and extrinsic elements involved in the process of intestinal homeostasis, injury, and repair, which fine-tune the balance between self-renewal and cell fate specification in ISCs. Deciphering the regulatory machinery that modulates stem cell fate would aid in the development of novel therapeutics that facilitate mucosal healing and restore epithelial barrier function.     

Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells

Intestinal stem cells, characterized by high Lgr5 expression, reside between Paneth cells at the small intestinal crypt base and divide every day. We have carried out fate mapping of individual stem cells by generating a multicolor Cre-reporter. As a population, Lgr5(hi) stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. We have collected short- and long-term clonal tracing data of individual Lgr5(hi) cells. These reveal that most Lgr5(hi) cell divisions occur symmetrically and do not support a model in which two daughter cells resulting from an Lgr5(hi) cell division adopt divergent fates (i.e., one Lgr5(hi) cell and one transit-amplifying [TA] cell per division). The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates. Quantitative analysis shows that stem cell turnover follows a pattern of neutral drift dynamics.     

Cdx2 determines the fate of postnatal intestinal endoderm

Knock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in the midgut. Conditional inactivation of Cdx2 in adult intestinal epithelium, as well as specifically in the Lgr5-positive stem cells, of adult mice allows long-term survival of the animals but fails to produce this phenotype. Instead, the endodermal cells exhibit cell-autonomous expression of gastric genes in an intestinal setting that is not accompanied by mesodermal expression of Barx1, which is necessary for gastric morphogenesis. Cdx2-negative endodermal cells also fail to express Sox2, a marker of gastric morphogenesis. Maturation of the stem cell niche thus appears to be associated with loss of ability to express positional information cues that are required for normal stomach development. Cdx2-negative intestinal crypts produce subsurface cystic vesicles, whereas untargeted crypts hypertrophy to later replace the surface epithelium. These observations are supported by studies involving inactivation of Cdx2 in intestinal crypts cultured in vitro. This abolishes their ability to form long-term growing intestinal organoids that differentiate into intestinal phenotypes. We conclude that expression of Cdx2 is essential for differentiation of gut stem cells into any of the intestinal cell types, but they maintain a degree of cell-autonomous plasticity that allows them to switch on a variety of gastric genes.     

Biased competition between Lgr5 intestinal stem cells driven by oncogenic mutation induces clonal expansion

The concept of ‘field cancerization’ describes the clonal expansion of genetically altered, but morphologically normal cells that predisposes a tissue to cancer development. Here, we demonstrate that biased stem cell competition in the mouse small intestine can initiate the expansion of such clones. We quantitatively analyze how the activation of oncogenic K-ras in individual Lgr5⁺ stem cells accelerates their cell division rate and creates a biased drift towards crypt clonality. K-ras mutant crypts then clonally expand within the epithelium through enhanced crypt fission, which distributes the existing Paneth cell niche over the two new crypts. Thus, an unequal competition between wild-type and mutant intestinal stem cells initiates a biased drift that leads to the clonal expansion of crypts carrying oncogenic mutations.     

BMP signaling in homeostasis,transformation and inflammatory response of intestinal epithelium

Intestine is the organ for food digestion, nutrient absorption and pathogen defense, in which processes intestinal epithelium plays a central role. Intestinal epithelium undergoes fast turnover, and its homeostasis is regulated by multiple signaling pathways, including Wnt, Notch, Hippo and BMP pathways. BMP signaling has been shown to negatively regulate self-renewal of Lgr5⁺ intestinal stem cells, constrains the expansion of intestinal epithelium, therefore attenuating colorectal cancer formation. BMPs and their receptors are expressed in both epithelial and mesenchymal cells, suggesting a two-way interaction between the mesenchyme and epithelium. In this review, we summarize the current understanding of the function of BMP signaling in homeostasis, cancerous transformation and inflammatory response of intestinal epithelium.     

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。     

Leucine-rich repeat-containing G-protein coupled receptor 5 enriched organoids under chemically-defined growth conditions

An organoid is a complex, multi-cell three-dimensional (3D) structure that contains tissue-specific cells. Epithelial stem cells, which are marked by leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), have the potential for self-renewal and expansion as organoids. However, in the case of intestinal organoids from Lgr5-EGFP-IRES-CreERT2 transgenic mice, in vitro expansion of the Lgr5 expression is limited in a culture condition supplemented with essential proteins, such as epidermal growth factor (E), noggin (N), and R-spondin 1 (R). In this study, we hypothesized that self-renewal of Lgr5+ stem cells in a 3D culture system can be stimulated by defined compounds (CHIR99021, Valproic acid, Y-27632, and A83-01). Our results demonstrated that dissociated single cells from organoids were organized into a 3D structure in the four compounds containing the ENR culture medium in a 3D and two-dimensional (2D) culture system. Moreover, the Lgr5 expression level of organoids from the ENR- and compound-containing media increased. Furthermore, the conversion of cultured Lgr5+ stem cells from 2D to 3D was confirmed. Therefore, defined compounds promote the expansion of Lgr5+ stem cells in organoids.