脫氧膽酸通過調(diào)節(jié)干細胞重編程因子OCT4與SOX2誘導食管鱗狀上皮腸化生
發(fā)布時間：2019-05-15 12:45
【摘要】：背景和目的:Barrett食管(Barrett’s esophagus,BE)是指食管下段鱗狀上皮被化生的柱狀上皮所取代的一種病理現(xiàn)象,目前普遍認為BE是食管腺癌(ECA)的主要癌前病變。近年來全世界尤其是西方國家食管腺癌及BE的發(fā)病率增加,BE的研究受到廣泛重視。Barrett食管的細胞來源尚不清楚,目前主要存在轉(zhuǎn)定型(Transcommitment)和轉(zhuǎn)分化(Transdifferentiation)兩類假設,但這些假設都因缺乏直接證據(jù)而不能完全讓人信服。近10年來,誘導多能干細胞的提出及運用使得轉(zhuǎn)分化學說越來越受重視。BE一直被認為是胃食管反流病(GERD)的并發(fā)癥,在胃食管反流人群中BE的檢出率高達10%-15%。結(jié)合之前有研究證實膽汁成分中非結(jié)合膽汁酸脫氧膽酸(DCA)是導致BE發(fā)生及癌變的重要成分,我們提出:DCA等刺激可能通過調(diào)節(jié)細胞重編程因子OCT4及SOX2的表達導致成熟食管鱗狀上皮細胞逆分化為具有部分分化潛能的細胞,再在環(huán)境因素的作用下轉(zhuǎn)分化形成柱狀上皮,進而形成BE。本課題的目的是研究脫氧膽酸對正常食管鱗狀上皮中OCT4及SOX2表達的影響,進一步探討其在BE發(fā)生中的作用及機制,為尋找BE診斷和治療的新靶點提供理論依據(jù)。方法:1.在臨床標本及通過胃全切+食管十二指腸吻合的方法構(gòu)建的大鼠單純膽汁反流模型中通過免疫組化方法檢測正常食管、食管炎及BE中OCT4、SOX2及腸型標記分子Cdx2、MUC2及鱗狀上皮標記分子p63表達水平;2.用200μmol/L的脫氧膽酸(DCA)分別處理Het-1A細胞0h、2h、4h、8h、12h,采用Real-time PCR、Western Blot等方法研究DCA對食管上皮細胞OCT4、SOX2、腸型標記分子Cdx2、MUC2及鱗狀上皮標記分子p63表達的影響;3.用慢病毒過表達及RNAi干擾的方法上調(diào)或下調(diào)Het-1A細胞中OCT4、SOX2的表達水平,觀察其對DCA誘導腸標記分子Cdx2、MUC2的影響以及OCT4、SOX2在DCA誘導食管腸上皮化生中的作用;4.觀察Het-1A細胞中OCT4表達調(diào)控對SOX2表達的影響及SOX2表達調(diào)控對OCT4表達的影響,探討食管鱗狀上皮細胞中OCT4與SOX2的相互關系。結(jié)果:1.人體標本中,正常食管上皮OCT4不表達,少量食管炎中OCT4呈微弱核表達,Barrett腺體及周邊鱗狀組織OCT4表達較正常食管及食管炎組織明顯升高。正常食管、食管炎、BE中SOX2表達逐漸下調(diào);2.臨床標本中,BE中腸型標記分子Cdx2、MUC2表達較正常食管、食管炎上調(diào),鱗狀上皮標記分子p63表達下調(diào);3.通過胃全切+食管十二指腸吻合術成功構(gòu)建了單純膽汁反流的大鼠模型成功誘導了BE形成。大鼠模型中OCT4、SOX2及腸型標記分子Cdx2、MUC2及鱗狀上皮標記分子p63表達與人體標本一致;4.體外實驗證實DCA處理能夠時間依賴性地上調(diào)鱗狀上皮細胞中OCT4的表達同時下調(diào)SOX2表達,并且DCA處理能誘導腸型標記分子Cdx2、MUC2的表達;5.干擾OCT4后腸型標記分子下調(diào)趨勢,OCT4干擾削弱了DCA處理對腸型標記分子的上調(diào)作用。單獨過表達OCT4并不足以誘導Cdx2、MUC2的表達;6.干擾掉SOX2后腸型標志Cdx2及MUC2上調(diào),并且對DCA誘導腸型分化表現(xiàn)出協(xié)同作用,過表達SOX2會導致腸型標志物表達下調(diào);7.干擾或過表達OCT4未影響SOX2表達變化,同樣干擾或過表達SOX2也未影響OCT4表達變化。結(jié)論:1.OCT4及SOX2可能在膽汁反流誘導BE形成的過程中起重要作用;2.大鼠模型中,單純膽汁反流能夠成功誘導食管炎及BE的形成;3.脫氧膽酸通過上調(diào)干細胞重編程因子OCT4及下調(diào)SOX2誘導成熟的鱗狀上皮轉(zhuǎn)分化形成腸型柱狀上皮;4.干擾或過表達OCT4并沒有影響SOX2表達變化,反之亦然,提示OCT4在食管上皮細胞轉(zhuǎn)分化過程中可能有其他的協(xié)同因子。
[Abstract]:Background and Objective: Barrett's esopagus (BE) is a kind of pathological phenomenon which is replaced by the columnar epithelium of the squamous epithelium of the lower segment of the esophagus. It is widely believed that BE is the main precancerous lesion of the esophageal adenocarcinoma (ECA). In recent years, the incidence of esophageal adenocarcinoma and BE in the world, especially in the western countries, has increased, and the research of BE has been widely paid attention to. The cell origin of Barrett's esophagus is not clear, and there are two types of hypotheses for transdifferentiation and transdifferentiation, but these assumptions are not entirely convincing for lack of direct evidence. In recent 10 years, the introduction and application of induced pluripotent stem cells have made the theory of transdifferentiation more and more attention. BE has been considered to be a complication of gastroesophageal reflux disease (GERD), with a detection rate of up to 10% to 15% in the gastroesophageal reflux population. In combination with previous studies, it was confirmed that the non-conjugated bile acid deoxycholic acid (DCA) in the bile component was an important component that led to the occurrence of BE and cancer, and we proposed: The stimulation of DCA and the like may lead to the reverse differentiation of mature esophageal squamous epithelial cells into cells with partial differentiation potential by regulating the expression of the cell reprogramming factors OCT4 and SOX2, and then to form the columnar epithelium under the action of environmental factors to form the BE. The purpose of this study is to study the effect of deoxycholic acid on the expression of OCT4 and SOX2 in the normal esophageal squamous epithelium, and to further explore the role and mechanism of deoxycholic acid in the occurrence of BE, and provide a theoretical basis for finding a new target for the diagnosis and treatment of BE. Method:1. The expression levels of the OCT4, SOX2 and intestinal type marker molecules Cdx2, MUC2 and squamous epithelial marker molecules p63 in the normal esophagus, the esophagitis and the BE were detected by the immunohistochemical method in the simple bile reflux model of the rat, which was constructed by the method of the full-incision of the stomach and the gastroduodenal anastomosis, and the expression level of the OCT4, SOX2 and the intestinal type marker molecules Cdx2, MUC2 and the squamous epithelium marker molecules p63 in the normal esophagus, the esophagitis and the BE is detected by an immunohistochemical method. The effects of DCA on the expression of human esophageal epithelial cell OCT4, SOX2, intestinal type marker, Cdx2, MUC2 and squamous epithelial marker p63 were studied by using 200. m u.mol/ L deoxycholic acid (DCA) respectively for 0 h,2 h,4 h,8 h and 12 h of Het-1A cells. The expression level of OCT4 and SOX2 in Het-1A cells was up-regulated or down-regulated by means of the overexpression of lentivirus and the interference of RNAi, and the effects of the expression of OCT4 and SOX2 on the induced intestinal metaplasia of the esophagus by DCA were observed. To observe the effect of the expression of OCT4 on the expression of SOX2 and the effect of the expression of SOX2 on the expression of OCT4 in Het-1A cells, the relationship between the expression of OCT4 and SOX2 in the squamous cell of the esophagus was discussed. Results:1. In the body specimen, the normal esophageal epithelium OCT4 was not expressed, and the OCT4 in the small amount of esophagitis showed a weak nuclear expression, and the expression of the Barrett's gland and the peripheral squamous cell OCT4 was higher than that of the normal esophageal and esophagitis. The expression of SOX2 in the normal esophagus, esophagitis and BE gradually decreased;2. In the clinical specimen, the expression of the intestinal type marker molecule Cdx2 and MUC2 in the BE is lower than that of the normal esophageal and esophagitis, and the expression of the squamous epithelial marker molecule p63 is down-regulated; and 3. The formation of BE was successfully induced by the successful construction of the rat model of simple bile reflux by the complete resection of the stomach and the esophagogastroduodenal anastomosis. In the rat model, the expression of OCT4, SOX2 and the intestinal type marker molecules Cdx2, MUC2 and squamous epithelium marker p63 was consistent with the human specimen;4. In vitro, the expression of the expression of the OCT4 in the squamous epithelial cells can be up-regulated in time-dependent manner, and the expression of the SOX2 is down-regulated, and the DCA treatment can induce the expression of the intestinal type marker molecules Cdx2 and MUC2. The down-regulation of the intestinal type marker molecules after interfering with OCT4, and the interference of OCT4, weakened the up-regulation of the DCA treatment on the intestinal type marker. The expression of OCT4 alone was not sufficient to induce expression of Cdx2, MUC2;6. The expression of the expression of SOX2 could result in a down-regulation of the expression of intestinal type markers, and the expression of SOX2 could result in a down-regulation of the expression of intestinal type markers. The interference or overexpression of OCT4 did not affect the expression of SOX2, and the same interference or overexpression of SOX2 did not affect the expression of OCT4. Conclusion:1. OCT4 and SOX2 may play an important role in the formation of bile reflux-induced BE. In the rat model, simple bile reflux can induce the formation of esophagitis and BE; Deoxycholic acid induced mature squamous epithelium by up-regulation of stem cell reprogramming factor OCT4 and down-regulation of SOX2 to form an intestinal columnar epithelium;4. The interference or overexpression of OCT4 does not affect the expression of SOX2 and vice versa, suggesting that OCT4 may have other co-factors in the transdifferentiation of esophageal epithelial cells.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R571
本文編號：2477516
[Abstract]:Background and Objective: Barrett's esopagus (BE) is a kind of pathological phenomenon which is replaced by the columnar epithelium of the squamous epithelium of the lower segment of the esophagus. It is widely believed that BE is the main precancerous lesion of the esophageal adenocarcinoma (ECA). In recent years, the incidence of esophageal adenocarcinoma and BE in the world, especially in the western countries, has increased, and the research of BE has been widely paid attention to. The cell origin of Barrett's esophagus is not clear, and there are two types of hypotheses for transdifferentiation and transdifferentiation, but these assumptions are not entirely convincing for lack of direct evidence. In recent 10 years, the introduction and application of induced pluripotent stem cells have made the theory of transdifferentiation more and more attention. BE has been considered to be a complication of gastroesophageal reflux disease (GERD), with a detection rate of up to 10% to 15% in the gastroesophageal reflux population. In combination with previous studies, it was confirmed that the non-conjugated bile acid deoxycholic acid (DCA) in the bile component was an important component that led to the occurrence of BE and cancer, and we proposed: The stimulation of DCA and the like may lead to the reverse differentiation of mature esophageal squamous epithelial cells into cells with partial differentiation potential by regulating the expression of the cell reprogramming factors OCT4 and SOX2, and then to form the columnar epithelium under the action of environmental factors to form the BE. The purpose of this study is to study the effect of deoxycholic acid on the expression of OCT4 and SOX2 in the normal esophageal squamous epithelium, and to further explore the role and mechanism of deoxycholic acid in the occurrence of BE, and provide a theoretical basis for finding a new target for the diagnosis and treatment of BE. Method:1. The expression levels of the OCT4, SOX2 and intestinal type marker molecules Cdx2, MUC2 and squamous epithelial marker molecules p63 in the normal esophagus, the esophagitis and the BE were detected by the immunohistochemical method in the simple bile reflux model of the rat, which was constructed by the method of the full-incision of the stomach and the gastroduodenal anastomosis, and the expression level of the OCT4, SOX2 and the intestinal type marker molecules Cdx2, MUC2 and the squamous epithelium marker molecules p63 in the normal esophagus, the esophagitis and the BE is detected by an immunohistochemical method. The effects of DCA on the expression of human esophageal epithelial cell OCT4, SOX2, intestinal type marker, Cdx2, MUC2 and squamous epithelial marker p63 were studied by using 200. m u.mol/ L deoxycholic acid (DCA) respectively for 0 h,2 h,4 h,8 h and 12 h of Het-1A cells. The expression level of OCT4 and SOX2 in Het-1A cells was up-regulated or down-regulated by means of the overexpression of lentivirus and the interference of RNAi, and the effects of the expression of OCT4 and SOX2 on the induced intestinal metaplasia of the esophagus by DCA were observed. To observe the effect of the expression of OCT4 on the expression of SOX2 and the effect of the expression of SOX2 on the expression of OCT4 in Het-1A cells, the relationship between the expression of OCT4 and SOX2 in the squamous cell of the esophagus was discussed. Results:1. In the body specimen, the normal esophageal epithelium OCT4 was not expressed, and the OCT4 in the small amount of esophagitis showed a weak nuclear expression, and the expression of the Barrett's gland and the peripheral squamous cell OCT4 was higher than that of the normal esophageal and esophagitis. The expression of SOX2 in the normal esophagus, esophagitis and BE gradually decreased;2. In the clinical specimen, the expression of the intestinal type marker molecule Cdx2 and MUC2 in the BE is lower than that of the normal esophageal and esophagitis, and the expression of the squamous epithelial marker molecule p63 is down-regulated; and 3. The formation of BE was successfully induced by the successful construction of the rat model of simple bile reflux by the complete resection of the stomach and the esophagogastroduodenal anastomosis. In the rat model, the expression of OCT4, SOX2 and the intestinal type marker molecules Cdx2, MUC2 and squamous epithelium marker p63 was consistent with the human specimen;4. In vitro, the expression of the expression of the OCT4 in the squamous epithelial cells can be up-regulated in time-dependent manner, and the expression of the SOX2 is down-regulated, and the DCA treatment can induce the expression of the intestinal type marker molecules Cdx2 and MUC2. The down-regulation of the intestinal type marker molecules after interfering with OCT4, and the interference of OCT4, weakened the up-regulation of the DCA treatment on the intestinal type marker. The expression of OCT4 alone was not sufficient to induce expression of Cdx2, MUC2;6. The expression of the expression of SOX2 could result in a down-regulation of the expression of intestinal type markers, and the expression of SOX2 could result in a down-regulation of the expression of intestinal type markers. The interference or overexpression of OCT4 did not affect the expression of SOX2, and the same interference or overexpression of SOX2 did not affect the expression of OCT4. Conclusion:1. OCT4 and SOX2 may play an important role in the formation of bile reflux-induced BE. In the rat model, simple bile reflux can induce the formation of esophagitis and BE; Deoxycholic acid induced mature squamous epithelium by up-regulation of stem cell reprogramming factor OCT4 and down-regulation of SOX2 to form an intestinal columnar epithelium;4. The interference or overexpression of OCT4 does not affect the expression of SOX2 and vice versa, suggesting that OCT4 may have other co-factors in the transdifferentiation of esophageal epithelial cells.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R571
【參考文獻】
相關期刊論文 前2條
1 趙晶京;房殿春;肖桃元;彭貴勇;章容;徐承平;于冬梅;;Barrett食管上皮的微細結(jié)構(gòu)及意義[J];第三軍醫(yī)大學學報;2009年16期
2 Richard M Peek;Steven M Levine;David T Pride;Martin J Blaser;;Bacterial biota in reflux esophagitis and Barrett's esophagus[J];World Journal of Gastroenterology;2005年46期
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