本文選題：KLF5 + 介導(dǎo)��； 參考：《第三軍醫(yī)大學(xué)》2016年碩士論文

【摘要】：背景與目的:Barrett食管(Barrett’s esophagus,簡稱BE)是指遠端食管鱗狀上皮被化生的柱狀上皮所取代的病理現(xiàn)象,是食管腺癌重要的癌前病變。由于BE具有易癌變的特性,使其在臨床受到高度重視并成為研究食管腺癌的主要對象。雖然BE于19世紀50年代已在病理學(xué)上被定義、19世紀70年代已經(jīng)被認為是食管腺癌的形成因素,但對于引起B(yǎng)arrett食管的分子機制,我們的了解仍然非常有限。臨床觀察表明,多數(shù)食管腺癌的發(fā)生經(jīng)歷了胃食管反流(GERD)、BE至食管腺癌的系列演變過程。研究認為胃食管反流物的刺激導(dǎo)致BE的發(fā)生,胃酸與膽鹽是反流物的主要成分,以往研究認為胃酸與GERD、BE的嚴重程度密切相關(guān),而膽鹽在BE發(fā)生中的作用仍未引起足夠的重視。近期研究發(fā)現(xiàn),膽鹽在BE相關(guān)腺癌的發(fā)病過程中起重要作用,但膽鹽引起B(yǎng)E發(fā)生的機制仍未完全明了。KLF5(Krüppel-like factor 5)是一種鋅指蛋白,屬于Sp/KLF蛋白家族,高表達于腸道上皮細胞,是維持成人小腸的隱窩-絨毛軸所必需的因子,小腸絨毛的形成有賴于KLF5的調(diào)控;黏蛋白2(MUC2)大量表達于杯狀細胞,被認為是杯狀細胞的特異性標志物,MUC2也可在臨床病理診斷中作為Barrett食管杯狀細胞的分子標志;CDX2是一種核轉(zhuǎn)錄因子,在腸道上皮的生長、增殖以及分化過程中具有重要作用,它也是Barrett食管特征性表達因子之一;已有研究表明,膽酸能上調(diào)正常食管細胞系中腸化標志物CDX2和杯狀細胞標志物MUC2表達。小腸絨毛標志物VILLIN是一種肌動蛋白粘連蛋白,特異性表達于小腸絨毛刷狀緣;研究發(fā)現(xiàn)VILLIN在BE及食管腺癌病組織表達量較高。所以,MUC2、CDX2、VILLIN等腸道重要標志物都與Barrett食管的形成相關(guān),而KLF5可以促進小腸的生長發(fā)育、調(diào)控小腸絨毛的形成,與MUC2、CDX2及VILLIN等腸道標志物密切相關(guān);但KLF5是否通過調(diào)控上述腸道標志物介導(dǎo)BE組織形成,迄今尚無研究報道。本課題采集正常人群、臨床診斷食管炎、BE人群食管組織標本,并構(gòu)建大鼠食管炎及Barrett食管動物模型,進行免疫組化實驗,旨在探討KLF5是否與Barrett食管相關(guān);用DCA處理食管鱗狀上皮細胞株HET-1a細胞模擬體外反流實驗,探討KLF5是否參與腸化的形成過程;干擾及過表達正常食管細胞中KLF5,探討KLF5對MUC2、CDX2及VILLIN等腸化標志物的調(diào)節(jié)作用。揭示了在DCA誘導(dǎo)下KLF5可以通過調(diào)節(jié)MUC2、CDX2及VILLIN等因子的表達,導(dǎo)致食管鱗狀上皮細胞轉(zhuǎn)分化為柱狀上皮表型。研究方法:1.飼養(yǎng)Sprague-Dawley(SD)大鼠,并施以外科手術(shù)構(gòu)建膽酸反流模型,取其食管組織,制成免疫組化切片;2.收集第三軍醫(yī)大學(xué)第一附屬醫(yī)院2013年3月至2013年12月的反流性食管炎(RE)標本59例,短段BE標本21例;3.免疫組化檢測人和老鼠的食管鱗狀上皮、食管炎及BE組織中KLF5、MUC2、VILLIN、CDX2在組織中蛋白表達水平;4.培養(yǎng)人食管鱗狀上皮細胞(HET-1a),用濃度為200umol/L的去氧膽酸(DCA)按不同時間點處理HET-1a細胞,分別為0h、2h、4h、8h、12h;5.Real-time PCR(實時熒光定量PCR)、Western blot檢測DCA處理后,各時間點HET-1a細胞內(nèi)KLF5、VILLIN、MUC2及CDX2的m RNA及蛋白表達水平;6.下調(diào)KLF5后,Real-time PCR(實時熒光定量PCR)、Western blot檢測細胞中KLF5、MUC2、CDX2及VILLIN的m RNA及蛋白水平表達情況;7.過表達KLF5,采用Real-time PCR、Western blot檢測在細胞內(nèi)KLF5、VILLIN、MUC2及CDX2的m RNA及蛋白表達水平;8.免疫熒光檢測上述處理后HET-1a細胞內(nèi)KLF5、VILLIN、MUC2及CDX2的蛋白表達情況。結(jié)果:1.免疫組化結(jié)果顯示,在人類及大鼠BE食管上皮組織中KLF5、VILLIN、MUC2、及CDX2的表達呈強陽性,在食管炎中表達呈弱陽性或不表達,而在正常食管中表達呈陰性;2.HET-1a細胞經(jīng)DCA處理后,Real-time及Western blot檢驗發(fā)現(xiàn),與對照組相比隨著處理時間的延長,KLF5、VILLIN、MUC2、及CDX2的m RNA及蛋白水平的表達也呈上升趨勢;3.si RNA干擾細胞后,Real-time及Western blot檢驗發(fā)現(xiàn),與陰性對照組相比食管鱗狀上皮細胞中VILLIN、MUC2、及CDX2的m RNA及蛋白表達受到抑制,且對DCA誘導(dǎo)上述因子的表達也有明顯的抑制作用;4.慢病毒過表達KLF5后,Real-time及Western blot檢測發(fā)現(xiàn)腸道標志物VILLIN、MUC2、及CDX2的m RNA及蛋白與陰性對照相比表達也明顯增加。5.經(jīng)過上述1,2,3,4相同處理因素后,免疫熒光實驗檢測HET-1a細胞內(nèi)KLF5、MUC2、CDX2、VILLIN的蛋白表達趨勢,檢測結(jié)果與上述實驗結(jié)果一致。結(jié)論:1.KLF5在大鼠和人BE組織中的表達明顯高于食管炎癥以及正常組織,說明KLF5與Barrett食管相關(guān)。2.DCA處理HET-1a細胞后,隨著處理時間的延長,KLF5以及MUC2、CDX2以及VILLIN的腸道標志物的表達明顯增加,這在細胞實驗上進一步證明了KLF5與Barrett食管的形成相關(guān),且DCA能誘導(dǎo)KLF5、MUC2、CDX2、VILLIN的表達。3.抑制HET-1a細胞內(nèi)KLF5的表達,MUC2、CDX2以及VILLIN表達也明顯下調(diào),說明KLF5可能位于它們的上游,并能調(diào)控它們的表達。4.上調(diào)HET-1a細胞內(nèi)KLF5的表達發(fā)現(xiàn)MUC2、CDX2以及VILLIN表達明顯增高,說明KLF5能夠誘導(dǎo)上述因子的表達。以上發(fā)現(xiàn)提示,KLF5在DCA的介導(dǎo)下可誘導(dǎo)CDX2、MUC2、VILLIN等因子的表達,導(dǎo)致成熟的食管鱗狀上皮細胞轉(zhuǎn)分化為腸型柱狀上皮細胞,亦即BE上皮細胞。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R571

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