KLF5在DCA介导下诱导食管鳞状上皮向柱状上皮转分化的作用及机制研究

发布时间：2018-05-01 12:12

本文选题：KLF5 + 介导　；参考：《第三军医大学》2016年硕士论文

【摘要】：背景与目的:Barrett食管(Barrett’s esophagus,简称BE)是指远端食管鳞状上皮被化生的柱状上皮所取代的病理现象,是食管腺癌重要的癌前病变。由于BE具有易癌变的特性,使其在临床受到高度重视并成为研究食管腺癌的主要对象。虽然BE于19世纪50年代已在病理学上被定义、19世纪70年代已经被认为是食管腺癌的形成因素,但对于引起Barrett食管的分子机制,我们的了解仍然非常有限。临床观察表明,多数食管腺癌的发生经历了胃食管反流(GERD)、BE至食管腺癌的系列演变过程。研究认为胃食管反流物的刺激导致BE的发生,胃酸与胆盐是反流物的主要成分,以往研究认为胃酸与GERD、BE的严重程度密切相关,而胆盐在BE发生中的作用仍未引起足够的重视。近期研究发现,胆盐在BE相关腺癌的发病过程中起重要作用,但胆盐引起BE发生的机制仍未完全明了。KLF5(Krüppel-like factor 5)是一种锌指蛋白,属于Sp/KLF蛋白家族,高表达于肠道上皮细胞,是维持成人小肠的隐窝-绒毛轴所必需的因子,小肠绒毛的形成有赖于KLF5的调控;黏蛋白2(MUC2)大量表达于杯状细胞,被认为是杯状细胞的特异性标志物,MUC2也可在临床病理诊断中作为Barrett食管杯状细胞的分子标志;CDX2是一种核转录因子,在肠道上皮的生长、增殖以及分化过程中具有重要作用,它也是Barrett食管特征性表达因子之一;已有研究表明,胆酸能上调正常食管细胞系中肠化标志物CDX2和杯状细胞标志物MUC2表达。小肠绒毛标志物VILLIN是一种肌动蛋白粘连蛋白,特异性表达于小肠绒毛刷状缘;研究发现VILLIN在BE及食管腺癌病组织表达量较高。所以,MUC2、CDX2、VILLIN等肠道重要标志物都与Barrett食管的形成相关,而KLF5可以促进小肠的生长发育、调控小肠绒毛的形成,与MUC2、CDX2及VILLIN等肠道标志物密切相关;但KLF5是否通过调控上述肠道标志物介导BE组织形成,迄今尚无研究报道。本课题采集正常人群、临床诊断食管炎、BE人群食管组织标本,并构建大鼠食管炎及Barrett食管动物模型,进行免疫组化实验,旨在探讨KLF5是否与Barrett食管相关;用DCA处理食管鳞状上皮细胞株HET-1a细胞模拟体外反流实验,探讨KLF5是否参与肠化的形成过程;干扰及过表达正常食管细胞中KLF5,探讨KLF5对MUC2、CDX2及VILLIN等肠化标志物的调节作用。揭示了在DCA诱导下KLF5可以通过调节MUC2、CDX2及VILLIN等因子的表达,导致食管鳞状上皮细胞转分化为柱状上皮表型。研究方法:1.饲养Sprague-Dawley(SD)大鼠,并施以外科手术构建胆酸反流模型,取其食管组织,制成免疫组化切片;2.收集第三军医大学第一附属医院2013年3月至2013年12月的反流性食管炎(RE)标本59例,短段BE标本21例;3.免疫组化检测人和老鼠的食管鳞状上皮、食管炎及BE组织中KLF5、MUC2、VILLIN、CDX2在组织中蛋白表达水平;4.培养人食管鳞状上皮细胞(HET-1a),用浓度为200umol/L的去氧胆酸(DCA)按不同时间点处理HET-1a细胞,分别为0h、2h、4h、8h、12h;5.Real-time PCR(实时荧光定量PCR)、Western blot检测DCA处理后,各时间点HET-1a细胞内KLF5、VILLIN、MUC2及CDX2的m RNA及蛋白表达水平;6.下调KLF5后,Real-time PCR(实时荧光定量PCR)、Western blot检测细胞中KLF5、MUC2、CDX2及VILLIN的m RNA及蛋白水平表达情况;7.过表达KLF5,采用Real-time PCR、Western blot检测在细胞内KLF5、VILLIN、MUC2及CDX2的m RNA及蛋白表达水平;8.免疫荧光检测上述处理后HET-1a细胞内KLF5、VILLIN、MUC2及CDX2的蛋白表达情况。结果:1.免疫组化结果显示,在人类及大鼠BE食管上皮组织中KLF5、VILLIN、MUC2、及CDX2的表达呈强阳性,在食管炎中表达呈弱阳性或不表达,而在正常食管中表达呈阴性;2.HET-1a细胞经DCA处理后,Real-time及Western blot检验发现,与对照组相比随着处理时间的延长,KLF5、VILLIN、MUC2、及CDX2的m RNA及蛋白水平的表达也呈上升趋势;3.si RNA干扰细胞后,Real-time及Western blot检验发现,与阴性对照组相比食管鳞状上皮细胞中VILLIN、MUC2、及CDX2的m RNA及蛋白表达受到抑制,且对DCA诱导上述因子的表达也有明显的抑制作用;4.慢病毒过表达KLF5后,Real-time及Western blot检测发现肠道标志物VILLIN、MUC2、及CDX2的m RNA及蛋白与阴性对照相比表达也明显增加。5.经过上述1,2,3,4相同处理因素后,免疫荧光实验检测HET-1a细胞内KLF5、MUC2、CDX2、VILLIN的蛋白表达趋势,检测结果与上述实验结果一致。结论:1.KLF5在大鼠和人BE组织中的表达明显高于食管炎症以及正常组织,说明KLF5与Barrett食管相关。2.DCA处理HET-1a细胞后,随着处理时间的延长,KLF5以及MUC2、CDX2以及VILLIN的肠道标志物的表达明显增加,这在细胞实验上进一步证明了KLF5与Barrett食管的形成相关,且DCA能诱导KLF5、MUC2、CDX2、VILLIN的表达。3.抑制HET-1a细胞内KLF5的表达,MUC2、CDX2以及VILLIN表达也明显下调,说明KLF5可能位于它们的上游,并能调控它们的表达。4.上调HET-1a细胞内KLF5的表达发现MUC2、CDX2以及VILLIN表达明显增高,说明KLF5能够诱导上述因子的表达。以上发现提示,KLF5在DCA的介导下可诱导CDX2、MUC2、VILLIN等因子的表达,导致成熟的食管鳞状上皮细胞转分化为肠型柱状上皮细胞,亦即BE上皮细胞。
[Abstract]:Background and purpose: Barrett 's esophagus (BE) is a pathological phenomenon that is replaced by the columnar epithelium of the squamous epithelium of the distal esophagus. It is an important precancerous lesion of the carcinoma of the esophagus. Because BE has the characteristics of carcinogenesis, it is highly valued in the clinic and is the main object to study the adenocarcinoma of the esophagus. Although BE is 19 The 50s century has been defined pathologically, and in 1870s it has been considered as a factor in the formation of adenocarcinoma of the esophagus, but our understanding of the molecular mechanism of Barrett's esophagus is still very limited. Clinical observations show that most of the carcinomas of the esophagus undergo gastroesophageal reflux (GERD), and the evolution of BE to the adenocarcinoma of the esophagus. The study suggests that gastric and esophageal reflux stimulation leads to the occurrence of BE. Gastric acid and bile salts are the main components of reflux. Previous studies suggest that gastric acid is closely related to the severity of GERD and BE, while the role of bile salts in the occurrence of BE has not been paid enough attention. Recent studies have found that bile salts play an important role in the pathogenesis of BE related adenocarcinoma. But the mechanism of bile salt that causes BE is still not fully clear that.KLF5 (Kr u ppel-like factor 5) is a kind of zinc finger protein, which belongs to the Sp/KLF protein family and is highly expressed in the intestinal epithelial cells. It is a necessary factor to maintain the recess villi axis of the adult small intestine. The formation of small intestinal villi depends on the regulation of KLF5; mucin 2 (MUC2) is heavily expressed in the cup. Cells are considered to be a specific marker of goblet cells, and MUC2 can also be used as a molecular marker of Barrett's oesophagus goblet cells in clinicopathological diagnosis. CDX2 is a nuclear transcription factor that plays an important role in the growth, proliferation and differentiation of intestinal epithelium. It is also one of the characteristic expression factors of the Barrett esophagus. Cholic acid can increase the expression of intestinal marker CDX2 and goblet cell marker MUC2 in normal esophageal cell lines. Small intestinal villi marker VILLIN is a kind of actin adhesion protein, which is specifically expressed in the small intestinal fleece border. The study found that VILLIN in BE and esophageal adenocarcinoma tissue is high. Therefore, MUC2, CDX2, VILLIN and other important intestinal tract are important. The markers are related to the formation of Barrett's esophagus, and KLF5 can promote the growth and development of the small intestine and regulate the formation of small intestinal villi, which is closely related to the intestinal markers such as MUC2, CDX2 and VILLIN. But there is no research report on whether KLF5 has been mediated by the regulation of the above-mentioned intestinal markers to mediate the formation of BE tissue. Esophagitis, BE group of esophagus tissue specimens, and construct rat esophagitis and Barrett esophagus animal model, carry out immunohistochemistry experiment, aim to explore whether KLF5 is associated with Barrett esophagus; DCA treatment of esophageal squamous cell cell strain HET-1a cells simulated in vitro reflux experiment, explore whether KLF5 participates in the formation of intestinal metaplasia, interference and overexpression. KLF5 in normal esophageal cells, to explore the regulation of KLF5 on MUC2, CDX2 and VILLIN and other intestinal markers. It is revealed that KLF5 can regulate the expression of MUC2, CDX2, VILLIN and other factors induced by DCA, leading to the transformation of the squamous epithelial cells of the esophagus into a columnar epithelia. 1. The cholic acid reflux model was constructed and the esophagus tissue was constructed to make immunohistochemical sections. 2. the 59 cases of reflux esophagitis (RE) from the First Affiliated Hospital of Third Military Medical University from March 2013 to December 2013 were collected and 21 cases of short segment BE specimens were collected. 3. immunohistochemistry was used to detect the squamous epithelium of the esophagus, esophagitis and BE tissues in human and mice, and KLF5, MUC2, VILLIN, CDX2 in the tissues of BE. Protein expression level in tissue; 4. cultured human esophageal squamous epithelial cells (HET-1a), deoxycholic acid (DCA) with a concentration of 200umol/L, treated HET-1a cells at different time points, respectively, 0h, 2h, 4h, 8h, 12h; 5.Real-time PCR (real-time fluorescent quantitative PCR). M RNA and protein expression level; 6. after downregulation of KLF5, Real-time PCR (real-time fluorescent quantitative PCR), Western blot detection of KLF5, MUC2, CDX2 and VILLIN, and protein level expression; 7. The protein expression of KLF5, VILLIN, MUC2 and CDX2 in HET-1a cells after the treatment was detected. Results: 1. immunohistochemical results showed that the expression of KLF5, VILLIN, MUC2, and CDX2 were strongly positive in human and rat BE esophageal epithelium, and the expression in esophagitis was weak positive or non expression, but negative in the normal esophagus; 2.HET-1a thin. After DCA treatment, Real-time and Western blot test found that the expression of M RNA and protein levels of KLF5, VILLIN, MUC2, and CDX2 increased as compared with the control group. 2, the expression of M RNA and protein of CDX2 was inhibited and the expression of DCA induced the expression of the above factors was also inhibited. After the expression of KLF5 in 4. lentivirus, Real-time and Western blot detected the intestinal markers, VILLIN, MUC2, CDX2 m and protein and negative expression. The protein expression trend of KLF5, MUC2, CDX2, VILLIN in HET-1a cells was detected by immunofluorescence test. The results were in agreement with the experimental results. Conclusion: the expression of 1.KLF5 in rats and human BE tissues is significantly higher than that of the esophagitis and normal tissues. It shows that KLF5 and Barrett esophagus related.2.DCA are treated with HET-1a cells, with the treatment with the treatment. The expression of intestinal markers in KLF5 and MUC2, CDX2 and VILLIN increased significantly, which further demonstrated that KLF5 was associated with the formation of Barrett esophagus, and DCA could induce KLF5, MUC2, CDX2, and VILLIN expression. The expression of KLF5 in HET-1a cells can be regulated by their expression.4. and the expression of MUC2, CDX2 and VILLIN is obviously increased, indicating that KLF5 can induce the expression of the above factors. These findings suggest that KLF5 can induce the expression of CDX2, MUC2, VILLIN and other factors in the medium of DCA. The skin cells differentiate into intestinal columnar epithelial cells, that is, BE epithelial cells.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R571

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