脱氧胆酸通过调节干细胞重编程因子OCT4与SOX2诱导食管鳞状上皮肠化生
发布时间：2019-05-15 12:45
【摘要】：背景和目的:Barrett食管(Barrett’s esophagus,BE)是指食管下段鳞状上皮被化生的柱状上皮所取代的一种病理现象,目前普遍认为BE是食管腺癌(ECA)的主要癌前病变。近年来全世界尤其是西方国家食管腺癌及BE的发病率增加,BE的研究受到广泛重视。Barrett食管的细胞来源尚不清楚,目前主要存在转定型(Transcommitment)和转分化(Transdifferentiation)两类假设,但这些假设都因缺乏直接证据而不能完全让人信服。近10年来,诱导多能干细胞的提出及运用使得转分化学说越来越受重视。BE一直被认为是胃食管反流病(GERD)的并发症,在胃食管反流人群中BE的检出率高达10%-15%。结合之前有研究证实胆汁成分中非结合胆汁酸脱氧胆酸(DCA)是导致BE发生及癌变的重要成分,我们提出:DCA等刺激可能通过调节细胞重编程因子OCT4及SOX2的表达导致成熟食管鳞状上皮细胞逆分化为具有部分分化潜能的细胞,再在环境因素的作用下转分化形成柱状上皮,进而形成BE。本课题的目的是研究脱氧胆酸对正常食管鳞状上皮中OCT4及SOX2表达的影响,进一步探讨其在BE发生中的作用及机制,为寻找BE诊断和治疗的新靶点提供理论依据。方法:1.在临床标本及通过胃全切+食管十二指肠吻合的方法构建的大鼠单纯胆汁反流模型中通过免疫组化方法检测正常食管、食管炎及BE中OCT4、SOX2及肠型标记分子Cdx2、MUC2及鳞状上皮标记分子p63表达水平;2.用200μmol/L的脱氧胆酸(DCA)分别处理Het-1A细胞0h、2h、4h、8h、12h,采用Real-time PCR、Western Blot等方法研究DCA对食管上皮细胞OCT4、SOX2、肠型标记分子Cdx2、MUC2及鳞状上皮标记分子p63表达的影响;3.用慢病毒过表达及RNAi干扰的方法上调或下调Het-1A细胞中OCT4、SOX2的表达水平,观察其对DCA诱导肠标记分子Cdx2、MUC2的影响以及OCT4、SOX2在DCA诱导食管肠上皮化生中的作用;4.观察Het-1A细胞中OCT4表达调控对SOX2表达的影响及SOX2表达调控对OCT4表达的影响,探讨食管鳞状上皮细胞中OCT4与SOX2的相互关系。结果:1.人体标本中,正常食管上皮OCT4不表达,少量食管炎中OCT4呈微弱核表达,Barrett腺体及周边鳞状组织OCT4表达较正常食管及食管炎组织明显升高。正常食管、食管炎、BE中SOX2表达逐渐下调;2.临床标本中,BE中肠型标记分子Cdx2、MUC2表达较正常食管、食管炎上调,鳞状上皮标记分子p63表达下调;3.通过胃全切+食管十二指肠吻合术成功构建了单纯胆汁反流的大鼠模型成功诱导了BE形成。大鼠模型中OCT4、SOX2及肠型标记分子Cdx2、MUC2及鳞状上皮标记分子p63表达与人体标本一致;4.体外实验证实DCA处理能够时间依赖性地上调鳞状上皮细胞中OCT4的表达同时下调SOX2表达,并且DCA处理能诱导肠型标记分子Cdx2、MUC2的表达;5.干扰OCT4后肠型标记分子下调趋势,OCT4干扰削弱了DCA处理对肠型标记分子的上调作用。单独过表达OCT4并不足以诱导Cdx2、MUC2的表达;6.干扰掉SOX2后肠型标志Cdx2及MUC2上调,并且对DCA诱导肠型分化表现出协同作用,过表达SOX2会导致肠型标志物表达下调;7.干扰或过表达OCT4未影响SOX2表达变化,同样干扰或过表达SOX2也未影响OCT4表达变化。结论:1.OCT4及SOX2可能在胆汁反流诱导BE形成的过程中起重要作用;2.大鼠模型中,单纯胆汁反流能够成功诱导食管炎及BE的形成;3.脱氧胆酸通过上调干细胞重编程因子OCT4及下调SOX2诱导成熟的鳞状上皮转分化形成肠型柱状上皮;4.干扰或过表达OCT4并没有影响SOX2表达变化,反之亦然,提示OCT4在食管上皮细胞转分化过程中可能有其他的协同因子。
[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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R571
本文编号：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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R571
【参考文献】
相关期刊论文 前2条
1 赵晶京;房殿春;肖桃元;彭贵勇;章容;徐承平;于冬梅;;Barrett食管上皮的微细结构及意义[J];第三军医大学学报;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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