發(fā)布時(shí)間：2018-01-01 20:40

  本文關(guān)鍵詞：成人小腸類器官體外培養(yǎng)體系的構(gòu)建及分化前后關(guān)鍵離子通道的表達(dá)與功能　出處：《南京大學(xué)》2015年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 小腸類器官 小腸干細(xì)胞 LGR5 WNT3A 離子轉(zhuǎn)運(yùn) Cl~-分泌 電中性NaCl吸收 CFTR NHE3 NKCC1 KCC

【摘要】：小腸類器官(enteroids)是一種衍生于小腸成體LGR5+干細(xì)胞的類器官模型。小腸類器官在細(xì)胞成分、組織架構(gòu)及特定功能等方面與小腸上皮組織高度相似,實(shí)現(xiàn)了在體外培養(yǎng)環(huán)境中對(duì)小腸上皮組織的復(fù)制。對(duì)比動(dòng)物實(shí)驗(yàn)?zāi)Ｐ秃统Ｒ?guī)細(xì)胞培養(yǎng)模型,小腸類器官具有多種優(yōu)勢(shì),特別是衍生于人體組織的小腸類器官,為在體外環(huán)境中了解人體內(nèi)小腸的真實(shí)狀況開啟了一個(gè)新的“窗口”。自Hans Clevers實(shí)驗(yàn)室于2009年報(bào)道成年小鼠小腸類器官、于2011年報(bào)道成人小腸類器官的建立方法以來(lái),小腸類器官迅速成為腸道研究領(lǐng)域的熱點(diǎn),為小腸生理與疾病的基礎(chǔ)研究、藥物的篩選和開發(fā)、再生醫(yī)學(xué)構(gòu)建人的個(gè)體化的“人工小腸”等,提供了一個(gè)全新的平臺(tái)。離子吸收和分泌是小腸的重要功能。在小腸上皮細(xì)胞表面,存在多種類型的離子通道,這些離子通道介導(dǎo)小腸上皮細(xì)胞的離子轉(zhuǎn)運(yùn),對(duì)維持機(jī)體水和電解質(zhì)的內(nèi)穩(wěn)態(tài)具有重要作用。Cl-分泌和電中性NaCl吸收是小腸上皮細(xì)胞最重要的離子轉(zhuǎn)運(yùn)過(guò)程,其相關(guān)離子通道的功能異常與某些小腸疾病(如腹瀉)的發(fā)生發(fā)展密切相關(guān)。使用人小腸類器官模型研究這些離子通道,可加深對(duì)人體小腸上皮細(xì)胞離子轉(zhuǎn)運(yùn)功能的認(rèn)識(shí),并為研究小腸生理功能、探討小腸疾病的病理生理機(jī)制、研發(fā)新型藥物(如止瀉藥/瀉藥)、以及再生醫(yī)學(xué)構(gòu)建人的個(gè)體化的“腸道芯片”和“人工小腸”等,提供基礎(chǔ)理論支持。本課題包括兩部分：第一部分是成人小腸類器官體外培養(yǎng)體系的構(gòu)建,目的是建立穩(wěn)定的成人小腸類器官原代培養(yǎng)和長(zhǎng)期擴(kuò)增體系,使應(yīng)用成人小腸類器官進(jìn)行相關(guān)研究成為可能；第二部分是成人小腸類器官分化前后關(guān)鍵離子通道的表達(dá)與功能,目的是了解成人小腸類器官的生理功能,重點(diǎn)研究參與Cr分泌和電中性NaCl吸收的離子通道,尋找新發(fā)現(xiàn)。第一部分成人小腸類器官體外培養(yǎng)體系的構(gòu)建一研究目的建立成人小腸類器官的原代培養(yǎng)和長(zhǎng)期擴(kuò)增體系；在此基礎(chǔ)上,構(gòu)建成人小腸類器官單層培養(yǎng)的方法,為相關(guān)功能學(xué)研究提供便利；驗(yàn)證成人小腸類器官分化方案的有效性,為使用未分化和分化的成人小腸類器官進(jìn)行對(duì)比研究提供證據(jù)支持。二研究方法(1)本研究通過(guò)消化內(nèi)鏡或外科手術(shù)獲取成人十二指腸組織標(biāo)本,參考Sato等和Foulke-Abel等報(bào)道的方法,建立成人小腸類器官的原代培養(yǎng)并進(jìn)行體外擴(kuò)增。(2)將成人小腸類器官接種于Transwell系統(tǒng),以建立單層培養(yǎng)體系,使用免疫熒光檢測(cè)細(xì)胞核、磷酸化埃茲蛋白、Na+-K+-ATP酶的定位,以評(píng)價(jià)細(xì)胞排列方式和細(xì)胞極性,動(dòng)態(tài)監(jiān)測(cè)跨膜電阻值的改變,以評(píng)估細(xì)胞間緊密連接的形成。(3)使用不含WNT3A的分化培養(yǎng)基,誘導(dǎo)成人小腸類器官分化,比較未分化與分化的成人小腸類器官在跨膜電阻值、堿性磷酸酶活性、LGR5+干細(xì)胞標(biāo)記物(LGR5、OLFM4)、潘氏細(xì)胞標(biāo)記物(LYZ)和終末分化小腸上皮細(xì)胞標(biāo)記物(SI)的rnRNA表達(dá)等方面的差異。三研究結(jié)果1 基于成人十二指腸組織標(biāo)本,本研究成功建立5例成人小腸類器官原代培養(yǎng)系。在體外培養(yǎng)體系中,5例成人小腸類器官培養(yǎng)均可連續(xù)傳代,并持續(xù)擴(kuò)增至少6個(gè)月。2 接種于Transwell系統(tǒng)后,成人小腸類器官包含的細(xì)胞呈單層、連續(xù)排列,磷酸化埃茲蛋白和Na+-K+-ATP酶的免疫熒光信號(hào)分別定位于細(xì)胞頂膜和基底側(cè)膜,跨膜電阻值逐漸升高,于接種后14天達(dá)到峰值。3 分化后,成人小腸類器官的跨膜電阻值和堿性磷酸酶活性顯著升高,LGR5、OLFM4、LYZ的mRN A表達(dá)顯著降低,SI的mRNA表達(dá)顯著升高。四結(jié)論本研究成功建立成人小腸類器官的原代培養(yǎng)和長(zhǎng)期擴(kuò)增體系；在此基礎(chǔ)上,使用Transwell系統(tǒng),成功建立成人小腸類器官的單層培養(yǎng)體系；證實(shí)使用不含WNT3A的分化培養(yǎng)基,可有效誘導(dǎo)成人小腸類器官分化。未分化和分化的成人小腸類器官分別具有小腸上皮組織隱窩和絨毛區(qū)域細(xì)胞的特征,可用于對(duì)比研究。第二部分成人小腸類器官分化前后關(guān)鍵離子通道的表達(dá)與功能一研究目的使用未分化和分化的成人小腸類器官,探討參與Cl-分泌和電中性NaCl吸收的關(guān)鍵離子通道的表達(dá)與功能。二研究方法對(duì)參與Cl-分泌的關(guān)鍵離子通道,包括細(xì)胞頂膜氯離子通道CFTR、ANO1、 ANO6、ANO10和細(xì)胞基底側(cè)膜離子通道NKCC1、KCC、KCNQ1/KCNE3、 KCNN4,以及參與電中性NaCl吸收的關(guān)鍵離子通道,包括細(xì)胞頂膜鈉離子／氫離子交換體NHE3、NHE2和陰離子交換體DRA、PAT1,分別進(jìn)行研究。(1)分別使用實(shí)時(shí)定量熒光PCR和免疫印跡,檢測(cè)各離子通道在未分化和分化成人小腸類器官中的mRNA和蛋白表達(dá)。(2)使用免疫熒光,檢測(cè)CFTR和NHE3在未分化和分化成人小腸類器官中的定位。(3)使用尤斯灌流室/電流鉗技術(shù),觀察毛喉素在未分化和分化成人小腸類器官中誘導(dǎo)Cl-分泌的效果,使用相關(guān)離子通道抑制劑觀察各離子通道在毛喉素引發(fā)的Cl-分泌過(guò)程中的作用。(4)使用細(xì)胞內(nèi)pH熒光探針BCECF-AM,在PTI熒光測(cè)量系統(tǒng)中測(cè)定未分化和分化成人小腸類器官的鈉離子/氫離子交換功能。三研究結(jié)果1 Cl-分泌相關(guān)離子通道的表達(dá)與功能1.1 細(xì)胞頂膜氯離子通道的蛋白表達(dá)在成人小腸類器官分化后無(wú)顯著改變,但細(xì)胞基底側(cè)膜相關(guān)離子通道的mRNA和蛋白表達(dá)顯著降低。免疫熒光顯示,CFTR在未分化和分化成人小腸類器官中均定位于細(xì)胞頂膜,信號(hào)強(qiáng)度無(wú)顯著差異。1.2在未分化和分化的成人小腸類器官中,毛喉素均可引發(fā)跨膜電位差和短路電流的升高。這一過(guò)程可被CFTR抑制劑CFTRiah-172完全抑制,被cAMP激活鉀離子通道抑制劑chromanol 293B部分抑制。1.3NKCC1抑制劑bumetanide可完全抑制毛喉素在未分化成人小腸類器官中引發(fā)的跨膜電位差和短路電流升高,但在分化的成人小腸類器官中無(wú)抑制作用。1.4在分化的成人小腸類器官中,對(duì)bumetanide無(wú)法抑制的毛喉素引發(fā)的跨膜電位差和短路電流改變,KCC抑制劑DIOA可完全抑制。2 電中性NaCl吸收相關(guān)離子通道的表達(dá)與功能2.1成人小腸類器官分化后,NHE3和NHE2的蛋白表達(dá)無(wú)顯著改變,但DRA和PAT1的mRNA表達(dá)顯著上升。免疫熒光顯示,NHE3在未分化和分化成人小腸類器官中均定位于細(xì)胞頂膜,信號(hào)強(qiáng)度無(wú)顯著差異。2.2未分化和分化的成人小腸類器官均具有良好的鈉離子/氫離子交換功能,二者無(wú)顯著差別。四結(jié)論本研究證實(shí),成人小腸類器官具有Cl-分泌和電中性NaCl吸收的結(jié)構(gòu)基礎(chǔ)與功能。(1)成人小腸類器官分化后,細(xì)胞頂膜氯離子通道的蛋白表達(dá)無(wú)顯著改變,但細(xì)胞基底側(cè)膜參與Cl-分泌的相關(guān)離子通道的mRNA和蛋白表達(dá)顯著降低。毛喉素可在成人小腸類器官中誘導(dǎo)Cl-分泌,這一過(guò)程涉及CFT R和cAMP激活鉀離子通道的參與。毛喉素在未分化和分化成人小腸類器官中誘導(dǎo)的Cl-分泌,分別依賴于NKCC1和KCC作為基底側(cè)氯離子轉(zhuǎn)運(yùn)通道。以上研究結(jié)果,為小腸上皮組織絨毛區(qū)域的細(xì)胞亦具有Cl-分泌功能的觀點(diǎn),提供了新的證據(jù),并且新發(fā)現(xiàn)KCC是絨毛區(qū)域細(xì)胞Cl-分泌過(guò)程中的重要離子通道。(2)成人小腸類器官分化后,鈉離子/氫離子交換體的蛋白表達(dá)無(wú)明顯改變,并且未分化和分化的成人小腸類器官具有相似的鈉離子／氫離子交換功能水平。以上研究結(jié)果,為小腸上皮組織隱窩區(qū)域的細(xì)胞亦具有電中性NaCl吸收功能的觀點(diǎn),提供了有力的新證據(jù)。
[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.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R656.7

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1 尹健一;成人小腸類器官體外培養(yǎng)體系的構(gòu)建及分化前后關(guān)鍵離子通道的表達(dá)與功能[D];南京大學(xué);2015年

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