本文關(guān)鍵詞：BMP4信號通路與KLF4在Barrett食管形成中的作用研究　出處：《第三軍醫(yī)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： BMP4 信號 通路 KLF4 Barrett 食管 形成 中的 作用 研究

【摘要】：背景目的Barrett食管是一種食管黏膜化生性病變,即遠(yuǎn)端正常食管鱗狀上皮被柱狀上皮取代的病變�，F(xiàn)在認(rèn)為,以膽汁酸反流為特征的反流性胃食管病是BE發(fā)生的主要風(fēng)險因素。反流的膽汁酸能夠?qū)е吗つど掀さ穆匝装Y和損傷,通過一系列分子生物學(xué)事件進(jìn)而導(dǎo)致正常食管上皮向BE的轉(zhuǎn)化,甚至最終導(dǎo)致食管腺癌的發(fā)生。然而,食管鱗狀上皮向柱狀上皮轉(zhuǎn)化過程中的分子事件目前仍不清楚。有學(xué)者通過基因表達(dá)的系列分析對比BE和正常食管黏膜中各基因的表達(dá)差異,試圖找出涉及正常食管上皮向BE的轉(zhuǎn)化的關(guān)鍵分子。骨形態(tài)發(fā)生蛋白4(Bone morphogenetic protein 4,BMP4)被檢測到僅在BE中高表達(dá)而不表達(dá)于正常食管黏膜。BMP4信號通路下游還包括BMP4受體,p-Smad 1/5/8和Smad 4。它們共同組成了BMP4信號通路。p-Smad 1/5/8蛋白能與Smad 4形成復(fù)合體轉(zhuǎn)移入核內(nèi),進(jìn)而參與調(diào)控某些目的基因。BMP4信號通路在細(xì)胞增殖,組織分化和胚胎發(fā)育中都起著至關(guān)重要的作用。一些研究表明BMP4信號通路也可能參與食管鱗狀上皮向BE的轉(zhuǎn)化過程。Krüppel樣鋅指轉(zhuǎn)錄因子4是一類鋅指蛋白類轉(zhuǎn)錄因子。其廣泛存在于各種器官組織中,參與調(diào)節(jié)細(xì)胞增殖,早起胚胎發(fā)育以及凋亡。KLF4在腫瘤形成和誘導(dǎo)多能干細(xì)胞中的作用被廣泛研究,作用非常重要�；谝陨涎芯拷Y(jié)果,我們猜想BMP4和KLF4可能共同參與促進(jìn)DCA誘導(dǎo)的食管黏膜化生。方法1.選取西南醫(yī)院2012年3-12月的71例消化內(nèi)科門診患者,簽署知情同意書后,按照正常和BE分組取活檢組織標(biāo)本,所有新鮮組織塊用10%福爾馬林固定,石蠟包埋,切成4μm薄片貼在載玻片。2.飼養(yǎng)SD大鼠,制作大鼠BE模型,模型完成后按照正常和BE分組取活檢組織標(biāo)本,所有新鮮組織塊用10%福爾馬林固定,石蠟包埋,切成4μm薄片貼在載玻片。3.制作正常食管組織和BE組織石蠟標(biāo)本,切片。免疫組化SP法染色檢測所有組織標(biāo)本中各蛋白表達(dá)水平,染色、封片、閱片處理。利用SPSS18.0軟件進(jìn)行對數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析,比較它們之間的關(guān)系。4.用BEBM培養(yǎng)基體外培養(yǎng)HET-1A、TE-1和OE33細(xì)胞。用200μmol/L的DCA,分別處理體外培養(yǎng)的細(xì)胞4、8、12h。5.以Gene Bank收錄的KLF4基因序列作為分析序列,設(shè)計(jì)si RNA干擾序列,將稀釋的羅氏轉(zhuǎn)染試劑與稀釋的si RNA輕輕混勻,室溫靜置20分鐘,以形成FAM-si RNA-轉(zhuǎn)染試劑混合物,對體外培養(yǎng)的細(xì)胞進(jìn)行干擾實(shí)驗(yàn)。6.載有卡那霉素固體培養(yǎng)基培養(yǎng)載有KLF4序列的細(xì)菌,接種環(huán)火上灼燒冷卻后蘸取質(zhì)粒菌液,按照“Z”字形劃板。半小時后將培養(yǎng)板倒置放在細(xì)菌孵箱14小時繼續(xù)培養(yǎng)。7.按照美國Sigma公司說明書,洗滌、離心、分離、濃縮、裂解、抽提質(zhì)粒。8.構(gòu)建LV-KLF4重組慢病毒,利用polybrene提高轉(zhuǎn)染效率,將稀釋液再與Enhanced Infection Solution混合均勻,加入進(jìn)體外培養(yǎng)的細(xì)胞中,對KLF4基因進(jìn)行過表達(dá)。熒光顯微鏡下觀察熒光表達(dá)情況。放入孵箱繼續(xù)培養(yǎng),等熒光穩(wěn)定后開始用嘌呤霉素篩選。9.使用添加外源性BMP4和Noggin對BMP4信號進(jìn)行激活或抑制,檢測下游分子的表達(dá)變化。10.利用特異性單克隆一抗孵育處理后的細(xì)胞,用載有熒光標(biāo)記的二抗結(jié)合一抗,再用激光共聚焦細(xì)胞免疫熒光檢測細(xì)胞中各基因的變化。11.使用q RT-PCR和Western blotting方法檢測各種處理組基因在m RNA和蛋白質(zhì)水平的表達(dá)變化。結(jié)果1.在人類標(biāo)本中,BE組織高表達(dá)BMP4,p-Smad1/5/8 and KLF4。我們用免疫組化SP法,染色證實(shí)了在人類BE標(biāo)本中,BMP4在正常食管組織當(dāng)中,陽性率9.1%,陽性染色主要集中在細(xì)胞質(zhì);p-Smad 1/5/8陽性率56.8%,陽性染色也主要集中在細(xì)胞核;KLF4陽性率61.4%,陽性染色也主要集中在細(xì)胞核。BE組織中,BMP4陽性率94%,陽性染色主要集中在細(xì)胞質(zhì);p-Smad 1/5/8陽性率96%,陽性染色也主要集中在細(xì)胞核;KLF4陽性率96%,陽性染色也主要集中在細(xì)胞核。BE組織中BMP4和p-Smad 1/5/8的陽性表達(dá)率均顯著高于正常食管組織,且差異具有顯著性(0.05)。2.在大鼠標(biāo)本中,BE組織高表達(dá)BMP4,p-Smad1/5/8 and KLF4。我們用免疫組化SP法,染色證實(shí)了大鼠BE組織中,正常食管組織BMP4陰性表達(dá);p-Smad 1/5/8陰性表達(dá)。BE組織中,BMP4陽性表達(dá),陽性染色主要集中在細(xì)胞質(zhì);p-Smad 1/5/8陽性表達(dá),陽性染色主要集中在細(xì)胞核;KLF4陽性表達(dá),陽性染色主要集中在細(xì)胞核。BE組織中BMP4、p-Smad 1/5/8和KLF4的陽性表達(dá)率均顯著高于正常食管組織。3.此外,在體外培養(yǎng)的細(xì)胞中,DCA上調(diào)了BMP4,KLF4,p-Smad 1/5/8,CDX2,MUC2 and MUC5ac的表達(dá)。我們還發(fā)現(xiàn)BMP4能夠上調(diào)KLF4,p-Smad 1/5/8,CDX2,MUC2 and MUC5ac的表達(dá),而Noggin則下調(diào)KLF4,p-Smad 1/5/8,CDX2,MUC2 and MUC5ac的表達(dá)。另外,經(jīng)過si RNA-KLF4干擾的細(xì)胞,CDX2,MUC2 and MUC5ac的表達(dá)下調(diào),此時BMP4不能夠再度刺激CDX2,MUC2 and MUC5ac的表達(dá)上調(diào)。同樣,經(jīng)過慢病毒過表達(dá)KLF4的細(xì)胞,CDX2,MUC2 and MUC5ac的表達(dá)上調(diào),此時Noggin不能夠再度引起CDX2,MUC2 and MUC5ac的表達(dá)下調(diào)。結(jié)論1.DCA促進(jìn)食管上皮細(xì)胞BMP4、p-Smad 1/5/8、KLF4以及CDX2,MUC2和MUC5ac的表達(dá)。2.外源性BMP4能激活BMP4信號通路,能夠上調(diào)p-Smad 1/5/8、KLF4以及CDX2,MUC2和MUC5ac的表達(dá)。3.KLF4的變化能夠影響CDX2,MUC2和MUC5ac的表達(dá)。4.DCA所誘導(dǎo)的細(xì)胞表型變化是首先通過激活BMP4信號通路,進(jìn)而引起KLF4上調(diào),最終促進(jìn)BE分子標(biāo)志CDX2,MUC2和MUC5ac的表達(dá)實(shí)現(xiàn)的。
[Abstract]:Background and objective Barrett's esophagus is a metaplastic esophageal mucosal lesion, the distal normal esophageal squamous epithelium was replaced by columnar epithelium lesions. Now that gastroesophageal reflux disease with bile acid reflux characteristics is the main risk factors of BE. Bile acid reflux can lead to chronic inflammation and injury the mucosal epithelium, through a series of molecular events leading to normal esophageal epithelial transformation to BE, and eventually lead to esophageal adenocarcinoma. However, esophageal squamous epithelium to columnar epithelium into molecular event in the process is still not clear. There are differences in gene expression between BE and normal esophageal mucosa in the analysis of scholars the gene expression of the series, trying to find the key to the normal esophageal epithelium involves molecular BE transformation. Bone morphogenetic protein 4 (Bone morphogenetic protein 4, BMP4) was detected only in BE high The expression but not expressed in normal esophageal mucosa downstream of.BMP4 signaling pathway including BMP4 receptor, p-Smad 1/5/8 and Smad 4. together constitute the signal pathway of BMP4.P-Smad 1/5/8 and Smad 4 protein can form a complex transfer into the nucleus, and some genes involved in the regulation of.BMP4 signaling pathway in cell proliferation, differentiation and embryonic development in all tissues the very important role. Some studies suggest that BMP4 signaling pathway may also be involved in esophageal squamous epithelium transformed into BE.Kr ppel like zinc finger transcription factor 4 is a class of zinc finger protein transcription factor. It widely exists in various organs and tissues, involved in the regulation of cell proliferation, apoptosis and early embryonic development of extensive research in the.KLF4 tumor formation and induced pluripotent stem cells in the role, the role is very important. Based on the above results, we think that BMP4 and KLF4 may be involved in promoting DCA Metaplasia of esophageal mucosa induced. Methods 1. cases of digestive 71 outpatients in Southwest Hospital from 2012 3-12 month, signed informed consent, in accordance with the normal group and BE biopsy specimens, all fresh tissue with 10% Faure Marin fixed, paraffin embedded, cut into 4 m slices with SD rats housed in slide.2., the rat models of BE were made, the model was completed in accordance with the normal packet and BE biopsy specimens, all fresh tissue with 10% Faure Marin fixed, paraffin embedded, cut into 4 m slices with paraffin specimens of normal esophageal tissue and BE tissue slides in.3. slices. SP immunohistochemical staining was used to detect all the protein expression level in tissue samples, staining, mounting, reading processing. The data were analyzed by using SPSS18.0 software, the relationship between them is compared with the.4. BEBM HET-1A culture medium in vitro, TE-1 and OE33 cells. With 200 mol/L of DCA, KLF4 gene sequences were treated in vitro 4,8,12h.5. cells with Gene Bank included as sequence analysis, design of Si RNA interference sequence, Si RNA transfection reagent dilution dilution and gently mix, standing at room temperature for 20 minutes to form a FAM-si RNA- transfection reagent mixture in vitro the cell interference experiment.6. containing kanamycin containing KLF4 sequence of solid culture medium for bacteria, the fire burning after cooling loop dipped into the plasmid bacteria, in accordance with the "Z" shape reticle. Half an hour after the culture plate upside down in bacteria incubation box 14 hours to continue training in accordance with the.7. Sigma company in the United States., washing, centrifugation, separation, concentration, pyrolysis, extraction of plasmid.8. to construct LV-KLF4 recombinant lentivirus, improve transfection efficiency by using Polybrene, Infection and Enhanced will be diluted Solution mixing, adding into in vitro The cells, the KLF4 gene was overexpressed. Observed under fluorescent microscope. The expression in the incubator to continue training, etc. after screening stable fluorescence.9. using exogenous BMP4 and Noggin activation or inhibition of BMP4 signaling by puromycin, detection of downstream molecules.10. expression by specific monoclonal antibody incubation of cells after treatment with fluorescent labeled two, carrying a combination of anti anti, expression of.11. gene by confocal immunofluorescence detection of cells using Q RT-PCR and Western blotting for the detection of various treatment group gene in M RNA and protein level. Results 1. in human specimens, BE the high expression of BMP4, p-Smad1/5/8 and KLF4., we used SP immunohistochemical method, BE staining confirmed in human specimens, BMP4 in normal esophageal tissues, the positive rate was 9.1%, the positive staining mainly concentrated In the cytoplasm; the positive rate of p-Smad 1/5/8 56.8%, positive staining was mainly concentrated in the nucleus; the positive rate of KLF4 was 61.4%, the positive staining was mainly concentrated in the nucleus in.BE tissues, the positive rate of BMP4 was 94%, the positive staining was mainly concentrated in the cytoplasm; the positive rate of p-Smad 1/5/8 96%, the positive staining was mainly concentrated in the nucleus; the positive rate of KLF4 96% the positive expression, positive staining mainly in the nucleus BMP4.BE organization and p-Smad 1/5/8 were significantly higher than those in normal esophageal tissues, and the difference was significant (0.05).2. in the mouse, BE high expression of BMP4, p-Smad1/5/8 and KLF4., we used SP immunohistochemical method, BE staining confirmed tissue in rats, expression of normal esophageal tissue BMP4 negative expression of p-Smad; 1/5/8 negative.BE, BMP4 positive expression, positive staining mainly in the cytoplasm; p-Smad 1/5/8 positive expression, positive staining was mainly concentrated in the cell Nucleus; the positive expression of KLF4, BMP4 positive staining mainly concentrated in the nucleus in.BE tissue, the positive expression of p-Smad 1/5/8 and KLF4 were significantly higher than those in normal esophageal tissues of.3. addition, in vitro cultured cells, DCA upregulation of BMP4, KLF4, p-Smad, 1/5/8, CDX2, and MUC5ac expression of MUC2. We also found that BMP4 upregulation of KLF4, p-Smad 1/5/8, CDX2 and MUC5ac, the expression of MUC2, Noggin p-Smad 1/5/8, down KLF4, CDX2, and MUC5ac expression of MUC2. In addition, after Si interference of RNA-KLF4 cells, CDX2, and expression of MUC2 MUC5ac, the BMP4 will not be able to re stimulate CDX2, upregulation of MUC2 and expression of MUC5ac. Similarly, after lentiviral overexpression of KLF4 cells, CDX2, and up-regulated MUC2 MUC5ac expression, while Noggin could not again cause CDX2, down regulate the expression of MUC2 and MUC5ac. Conclusion 1.DCA promotes esophageal epithelial cells BMP4, p-Smad 1/5/8, KLF4 and C DX2, MUC2 and MUC5ac.2. expression of exogenous BMP4 can activate the BMP4 signaling pathway, which can raise the expression of p-Smad 1/5/8, KLF4, CDX2, MUC2 and MUC5ac expression of.3.KLF4 could affect the CDX2 cell phenotype change induced by the expression of.4.DCA MUC2 and MUC5ac is the first by activating BMP4 signaling pathway and cause upregulation of KLF4, finally to promote the CDX2 logo BE, MUC2 expression and MUC5ac.

【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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4 本報記者 慕欣;從美國Barrett食管診治指南,借鑒什么[N];醫(yī)藥經(jīng)濟(jì)報;2010年

5 ;Barrett發(fā)誓戰(zhàn)勝技術(shù)衰退[N];網(wǎng)絡(luò)世界;2002年

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7 武警總醫(yī)院主任技師 李振華;認(rèn)識共聚焦激光顯微內(nèi)鏡[N];健康報;2008年

8 徐可樹 熊漢華 周霞;應(yīng)重視巴來特食管[N];家庭醫(yī)生報;2005年

9 阿勝　編譯;內(nèi)鏡新技術(shù),食管癌早知道[N];醫(yī)藥經(jīng)濟(jì)報;2010年

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6 王瑞華;選擇性環(huán)氧合酶-2抑制劑對實(shí)驗(yàn)性Barrett食管的作用研究[D];四川大學(xué);2006年

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8 趙晶京;Barrett食管粘膜微細(xì)結(jié)構(gòu)、粘蛋白表達(dá)及MTHFR基因多態(tài)性分析[D];第三軍醫(yī)大學(xué);2005年

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10 周超;ERK信號轉(zhuǎn)導(dǎo)通路在Barrett食管發(fā)生發(fā)展中的作用[D];河北醫(yī)科大學(xué);2011年

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