成人小肠类器官体外培养体系的构建及分化前后关键离子通道的表达与功能

发布时间：2018-01-01 20:40

本文关键词：成人小肠类器官体外培养体系的构建及分化前后关键离子通道的表达与功能　出处：《南京大学》2015年博士论文　论文类型：学位论文

【摘要】：小肠类器官(enteroids)是一种衍生于小肠成体LGR5+干细胞的类器官模型。小肠类器官在细胞成分、组织架构及特定功能等方面与小肠上皮组织高度相似,实现了在体外培养环境中对小肠上皮组织的复制。对比动物实验模型和常规细胞培养模型,小肠类器官具有多种优势,特别是衍生于人体组织的小肠类器官,为在体外环境中了解人体内小肠的真实状况开启了一个新的“窗口”。自Hans Clevers实验室于2009年报道成年小鼠小肠类器官、于2011年报道成人小肠类器官的建立方法以来,小肠类器官迅速成为肠道研究领域的热点,为小肠生理与疾病的基础研究、药物的筛选和开发、再生医学构建人的个体化的“人工小肠”等,提供了一个全新的平台。离子吸收和分泌是小肠的重要功能。在小肠上皮细胞表面,存在多种类型的离子通道,这些离子通道介导小肠上皮细胞的离子转运,对维持机体水和电解质的内稳态具有重要作用。Cl-分泌和电中性NaCl吸收是小肠上皮细胞最重要的离子转运过程,其相关离子通道的功能异常与某些小肠疾病(如腹泻)的发生发展密切相关。使用人小肠类器官模型研究这些离子通道,可加深对人体小肠上皮细胞离子转运功能的认识,并为研究小肠生理功能、探讨小肠疾病的病理生理机制、研发新型药物(如止泻药/泻药)、以及再生医学构建人的个体化的“肠道芯片”和“人工小肠”等,提供基础理论支持。本课题包括两部分：第一部分是成人小肠类器官体外培养体系的构建,目的是建立稳定的成人小肠类器官原代培养和长期扩增体系,使应用成人小肠类器官进行相关研究成为可能；第二部分是成人小肠类器官分化前后关键离子通道的表达与功能,目的是了解成人小肠类器官的生理功能,重点研究参与Cr分泌和电中性NaCl吸收的离子通道,寻找新发现。第一部分成人小肠类器官体外培养体系的构建一研究目的建立成人小肠类器官的原代培养和长期扩增体系；在此基础上,构建成人小肠类器官单层培养的方法,为相关功能学研究提供便利；验证成人小肠类器官分化方案的有效性,为使用未分化和分化的成人小肠类器官进行对比研究提供证据支持。二研究方法(1)本研究通过消化内镜或外科手术获取成人十二指肠组织标本,参考Sato等和Foulke-Abel等报道的方法,建立成人小肠类器官的原代培养并进行体外扩增。(2)将成人小肠类器官接种于Transwell系统,以建立单层培养体系,使用免疫荧光检测细胞核、磷酸化埃兹蛋白、Na+-K+-ATP酶的定位,以评价细胞排列方式和细胞极性,动态监测跨膜电阻值的改变,以评估细胞间紧密连接的形成。(3)使用不含WNT3A的分化培养基,诱导成人小肠类器官分化,比较未分化与分化的成人小肠类器官在跨膜电阻值、碱性磷酸酶活性、LGR5+干细胞标记物(LGR5、OLFM4)、潘氏细胞标记物(LYZ)和终末分化小肠上皮细胞标记物(SI)的rnRNA表达等方面的差异。三研究结果1 基于成人十二指肠组织标本,本研究成功建立5例成人小肠类器官原代培养系。在体外培养体系中,5例成人小肠类器官培养均可连续传代,并持续扩增至少6个月。2 接种于Transwell系统后,成人小肠类器官包含的细胞呈单层、连续排列,磷酸化埃兹蛋白和Na+-K+-ATP酶的免疫荧光信号分别定位于细胞顶膜和基底侧膜,跨膜电阻值逐渐升高,于接种后14天达到峰值。3 分化后,成人小肠类器官的跨膜电阻值和碱性磷酸酶活性显著升高,LGR5、OLFM4、LYZ的mRN A表达显著降低,SI的mRNA表达显著升高。四结论本研究成功建立成人小肠类器官的原代培养和长期扩增体系；在此基础上,使用Transwell系统,成功建立成人小肠类器官的单层培养体系；证实使用不含WNT3A的分化培养基,可有效诱导成人小肠类器官分化。未分化和分化的成人小肠类器官分别具有小肠上皮组织隐窝和绒毛区域细胞的特征,可用于对比研究。第二部分成人小肠类器官分化前后关键离子通道的表达与功能一研究目的使用未分化和分化的成人小肠类器官,探讨参与Cl-分泌和电中性NaCl吸收的关键离子通道的表达与功能。二研究方法对参与Cl-分泌的关键离子通道,包括细胞顶膜氯离子通道CFTR、ANO1、 ANO6、ANO10和细胞基底侧膜离子通道NKCC1、KCC、KCNQ1/KCNE3、 KCNN4,以及参与电中性NaCl吸收的关键离子通道,包括细胞顶膜钠离子／氢离子交换体NHE3、NHE2和阴离子交换体DRA、PAT1,分别进行研究。(1)分别使用实时定量荧光PCR和免疫印迹,检测各离子通道在未分化和分化成人小肠类器官中的mRNA和蛋白表达。(2)使用免疫荧光,检测CFTR和NHE3在未分化和分化成人小肠类器官中的定位。(3)使用尤斯灌流室/电流钳技术,观察毛喉素在未分化和分化成人小肠类器官中诱导Cl-分泌的效果,使用相关离子通道抑制剂观察各离子通道在毛喉素引发的Cl-分泌过程中的作用。(4)使用细胞内pH荧光探针BCECF-AM,在PTI荧光测量系统中测定未分化和分化成人小肠类器官的钠离子/氢离子交换功能。三研究结果1 Cl-分泌相关离子通道的表达与功能1.1 细胞顶膜氯离子通道的蛋白表达在成人小肠类器官分化后无显著改变,但细胞基底侧膜相关离子通道的mRNA和蛋白表达显著降低。免疫荧光显示,CFTR在未分化和分化成人小肠类器官中均定位于细胞顶膜,信号强度无显著差异。1.2在未分化和分化的成人小肠类器官中,毛喉素均可引发跨膜电位差和短路电流的升高。这一过程可被CFTR抑制剂CFTRiah-172完全抑制,被cAMP激活钾离子通道抑制剂chromanol 293B部分抑制。1.3NKCC1抑制剂bumetanide可完全抑制毛喉素在未分化成人小肠类器官中引发的跨膜电位差和短路电流升高,但在分化的成人小肠类器官中无抑制作用。1.4在分化的成人小肠类器官中,对bumetanide无法抑制的毛喉素引发的跨膜电位差和短路电流改变,KCC抑制剂DIOA可完全抑制。2 电中性NaCl吸收相关离子通道的表达与功能2.1成人小肠类器官分化后,NHE3和NHE2的蛋白表达无显著改变,但DRA和PAT1的mRNA表达显著上升。免疫荧光显示,NHE3在未分化和分化成人小肠类器官中均定位于细胞顶膜,信号强度无显著差异。2.2未分化和分化的成人小肠类器官均具有良好的钠离子/氢离子交换功能,二者无显著差别。四结论本研究证实,成人小肠类器官具有Cl-分泌和电中性NaCl吸收的结构基础与功能。(1)成人小肠类器官分化后,细胞顶膜氯离子通道的蛋白表达无显著改变,但细胞基底侧膜参与Cl-分泌的相关离子通道的mRNA和蛋白表达显著降低。毛喉素可在成人小肠类器官中诱导Cl-分泌,这一过程涉及CFT R和cAMP激活钾离子通道的参与。毛喉素在未分化和分化成人小肠类器官中诱导的Cl-分泌,分别依赖于NKCC1和KCC作为基底侧氯离子转运通道。以上研究结果,为小肠上皮组织绒毛区域的细胞亦具有Cl-分泌功能的观点,提供了新的证据,并且新发现KCC是绒毛区域细胞Cl-分泌过程中的重要离子通道。(2)成人小肠类器官分化后,钠离子/氢离子交换体的蛋白表达无明显改变,并且未分化和分化的成人小肠类器官具有相似的钠离子／氢离子交换功能水平。以上研究结果,为小肠上皮组织隐窝区域的细胞亦具有电中性NaCl吸收功能的观点,提供了有力的新证据。
[Abstract]:Intestinal organoid (enteroids) is a derivative of adult stem cells in the small intestine LGR5+ class organ model. The small intestine organ in cell composition, organizational structure and specific function and intestinal epithelial tissue was highly similar to that achieved in the replication of intestinal epithelial tissue in vitro. Comparison of experimental animal model and conventional cells the small intestine organ culture model has many advantages, especially in the small intestine organs derived from human tissue, and opened a new "window" in order to understand the true state of the human body in small intestine in vitro environment. Since the Hans Clevers laboratory in 2009 reported adult mouse intestinal organoid method established in 2011 reported adult small intestine the organ has intestinal organs quickly become a hot research field for the gut, intestinal physiology and disease research, drug screening and development, construction of regenerative medicine The individual "artificial intestinal", provides a new platform. The ion absorption and secretion is an important function of small intestine. In the intestinal epithelial cells, there are many types of ion channels, ion transport in these ion channels mediated by intestinal epithelial cells, to maintain the homeostasis of body water and electrolyte with the important role of.Cl- secretion and absorption of NaCl is electrically neutral ion transport in intestinal epithelial cells is the most important process, the abnormal function of ion channels and some intestinal diseases (such as diarrhea) is closely related to the occurrence and development of these ion channels. The use of the small intestine organ model, can deepen the understanding of human intestinal epithelial ion transport function the study of the small intestine and physiological function, to explore the pathophysiological mechanism of intestinal diseases, new drugs (such as antidiarrheal / laxatives) and regenerative medicine, construct the individual" The intestinal chip "and" artificial intestinal ", to provide a theoretical basis. This paper consists of two parts: the first part is the construction of the training system of small intestinal organs in vitro, the purpose is to establish a stable class of small intestinal organ cultured and long-term amplification system, the application of human intestinal organoid related research possible; the second part is the expression and function of the small intestine before and after adult organ differentiation key ion channels, to understand the physiological function of small intestine of adult organs, focusing on participation in the secretion of Cr and neutral NaCl absorption of ion channels, looking for new discoveries. Construction and long-term amplification system primary research objective to establish the culture system of small intestinal organs the first part of the small intestine of adult organs cultured in vitro; on this basis, the construction method of small intestinal organoid monolayer culture, studies provided for related functions For convenience; validation of adult small intestine organ differentiation scheme, for the use of undifferentiated and differentiated adult intestinal organoid comparative study provide evidence to support the. Two research methods (1) the acquisition of adult duodenal tissue through the digestive endoscopy or surgery, referred to Sato and Foulke-Abel reported the establishment of adult the small intestine organ cultured and amplified in vitro. (2) the class of adult small intestine inoculated in Transwell organ system, to establish a monolayer culture system, using immunofluorescence nucleus, phosphorylation of ezrin, Na+-K+-ATP enzyme, to evaluate cell arrangement and cell polarity, dynamic monitoring of membrane resistance in order to evaluate the change of intercellular tight junction formation. (3) use the differentiation medium without WNT3A, induced small intestinal organoid differentiation, relatively undifferentiated and differentiated adult small Intestinal type organs in transmembrane resistance, alkaline phosphatase activity, LGR5+ stem cell markers (LGR5, OLFM4), Paneth cell marker (LYZ) and terminal differentiation of intestinal epithelial cell marker (SI) between the rnRNA expression and so on. Three the results of 1 adult duodenal tissue samples based on this research established in 5 cases of small intestinal organoid. Primary cultured in vitro system, 5 cases of small intestinal organoid culture can be continuously passaged, amplified and sustained for at least 6 months after inoculation of.2 to Transwell system, including the cell organs of adult small intestine was single, continuous arrangement, immunofluorescence signal phosphorylation of Ezrin and the Na+-K+-ATP enzyme were located in the cells of apical and basolateral membrane, membrane resistance gradually increased to peak.3 differentiation reached 14 days after inoculation, the transepithelial electrical resistance of small intestinal organs and alkaline phosphatase activity increased significantly LGR5, OLFM4, mRN, A significantly decreased the expression of LYZ, SI mRNA expression was significantly increased. Conclusion four and long-term primary amplification system this study successfully established small intestinal organoid culture; on this basis, the use of the Transwell system, successfully established small intestinal organs Dan Cengpei raising system; use the differentiation medium is not confirmed with WNT3A, can effectively induce adult small intestine organ differentiation. The characteristics of undifferentiated and differentiated adult intestinal organs respectively with intestinal epithelial crypts and villi tissue region of the cell, can be used to study the expression and function of the second part. The adult small intestinal organoid differentiation before and after key ion channel research purpose using an adult small class organogenesis and differentiation, to investigate the expression and function of Cl- in neutral NaCl secretion and absorption of key ion channels. Two research methods to participate in key Cl- secretion from The channel, including cell apical membrane chloride ion channel CFTR, ANO1, ANO6, ANO10 and ion channels in the basolateral membrane of NKCC1 cells, KCC, KCNQ1/KCNE3, KCNN4, and channel key ion neutral NaCl absorption, including cell apical membrane sodium / hydrogen ion exchange of NHE3, NHE2 and anion exchange DRA, PAT1. Were studied. (1) using real-time quantitative PCR and Western blot, mRNA and protein expression in undifferentiated and differentiated organs in adult small intestine detected by ion channels. (2) using immunofluorescence detection of CFTR and NHE3 in the undifferentiated and differentiated positioning of adult small intestine class organs. (3) clamp technique using Ussing chamber / current, observe the forskolin induced Cl- secretion in undifferentiated and differentiated adult intestinal organs, associated with the use of ion channel inhibitors to observe the ion channels in forskolin induced Cl- secretion in the process (4). The use of intracellular pH fluorescence probe BCECF-AM, PTI fluorescence measurement system of sodium ion / determination of undifferentiated and differentiated adult intestinal organoid hydrogen ion exchange function. Three the results of expression and function of 1 Cl- secretion related ion channels in the 1.1 cell apical membrane chloride channel protein expression in the small intestine of adult organs after no significant differentiation change, but the expression of mRNA and protein in the cell basolateral membrane ion channel was significantly reduced. Immunofluorescence showed that CFTR was localized in the apical membrane of the cells in undifferentiated and differentiated adult intestinal organs, the signal intensity of.1.2 had no significant difference in adult intestinal organoid undifferentiated and differentiated, forskolin can lead to increased the transmembrane potential difference and short-circuit current. This process can be CFTR inhibitor CFTRiah-172 completely inhibited by cAMP activated potassium channel inhibitor chromanol 293B inhibitor bum inhibited.1.3NKCC1 Etanide completely inhibited forskolin in undifferentiated adult intestinal organoid transmembrane potential caused by the poor and short circuit current increases, but no inhibition of.1.4 in adult small intestine organ differentiation in adult small intestine organ differentiation, the transmembrane potential of bumetanide can not inhibit the forskolin induced differential and short circuit the current change, KCC inhibitor DIOA could completely inhibit the expression and function of.2 neutral NaCl absorption related ion channels in 2.1 adult small intestine organ differentiation, NHE3 and NHE2 protein expression had no significant change, but DRA and PAT1 mRNA expression increased significantly. Immunofluorescence showed that NHE3 was localized in the apical membrane of the cells in the undifferentiated and differentiation of adult small intestine organs in adult intestinal organoid signal intensity had no significant difference between undifferentiated and differentiated.2.2 with sodium ion / good hydrogen ion exchange function, no significant difference between the two and four conclusions of this research. Study confirmed that the adult small intestine organs with Cl- secretion and neutral NaCl absorption structure and function. (1) of small intestinal organs after differentiation, cell apical membrane chloride ion channel protein expression did not change significantly, but the expression of mRNA and protein of cells involved in the basolateral membrane ion channel Cl- secretion was significantly reduced. Forskolin in the small intestine of adult organs induced the secretion of Cl-, which involves the CFT R and cAMP activated potassium channels involved. Forskolin in undifferentiated and differentiated adult intestinal organoid induced the secretion of Cl-, NKCC1 and KCC were dependent on the basal side of chloride transport. The above research the results for the intestinal epithelial villi region of the cell also has Cl- secretion function, provide new evidence and new found that KCC is a regional villi Cl- secretory cell important ion channels in the process. (2) of small intestinal organs After differentiation, sodium / hydrogen ion exchanger protein expression had no obvious change, small intestinal organs and undifferentiated and differentiated with sodium / hydrogen ion exchange function of similar level. The above results for intestinal epithelial cell organization recess region also has NaCl absorption function in electric view provides powerful new evidence.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R656.7

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