BMP4信号通路与KLF4在Barrett食管形成中的作用研究

发布时间：2018-01-01 05:23

本文关键词：BMP4信号通路与KLF4在Barrett食管形成中的作用研究　出处：《第三军医大学》2016年硕士论文　论文类型：学位论文

【摘要】：背景目的Barrett食管是一种食管黏膜化生性病变,即远端正常食管鳞状上皮被柱状上皮取代的病变。现在认为,以胆汁酸反流为特征的反流性胃食管病是BE发生的主要风险因素。反流的胆汁酸能够导致黏膜上皮的慢性炎症和损伤,通过一系列分子生物学事件进而导致正常食管上皮向BE的转化,甚至最终导致食管腺癌的发生。然而,食管鳞状上皮向柱状上皮转化过程中的分子事件目前仍不清楚。有学者通过基因表达的系列分析对比BE和正常食管黏膜中各基因的表达差异,试图找出涉及正常食管上皮向BE的转化的关键分子。骨形态发生蛋白4(Bone morphogenetic protein 4,BMP4)被检测到仅在BE中高表达而不表达于正常食管黏膜。BMP4信号通路下游还包括BMP4受体,p-Smad 1/5/8和Smad 4。它们共同组成了BMP4信号通路。p-Smad 1/5/8蛋白能与Smad 4形成复合体转移入核内,进而参与调控某些目的基因。BMP4信号通路在细胞增殖,组织分化和胚胎发育中都起着至关重要的作用。一些研究表明BMP4信号通路也可能参与食管鳞状上皮向BE的转化过程。Krüppel样锌指转录因子4是一类锌指蛋白类转录因子。其广泛存在于各种器官组织中,参与调节细胞增殖,早起胚胎发育以及凋亡。KLF4在肿瘤形成和诱导多能干细胞中的作用被广泛研究,作用非常重要。基于以上研究结果,我们猜想BMP4和KLF4可能共同参与促进DCA诱导的食管黏膜化生。方法1.选取西南医院2012年3-12月的71例消化内科门诊患者,签署知情同意书后,按照正常和BE分组取活检组织标本,所有新鲜组织块用10%福尔马林固定,石蜡包埋,切成4μm薄片贴在载玻片。2.饲养SD大鼠,制作大鼠BE模型,模型完成后按照正常和BE分组取活检组织标本,所有新鲜组织块用10%福尔马林固定,石蜡包埋,切成4μm薄片贴在载玻片。3.制作正常食管组织和BE组织石蜡标本,切片。免疫组化SP法染色检测所有组织标本中各蛋白表达水平,染色、封片、阅片处理。利用SPSS18.0软件进行对数据进行统计学分析,比较它们之间的关系。4.用BEBM培养基体外培养HET-1A、TE-1和OE33细胞。用200μmol/L的DCA,分别处理体外培养的细胞4、8、12h。5.以Gene Bank收录的KLF4基因序列作为分析序列,设计si RNA干扰序列,将稀释的罗氏转染试剂与稀释的si RNA轻轻混匀,室温静置20分钟,以形成FAM-si RNA-转染试剂混合物,对体外培养的细胞进行干扰实验。6.载有卡那霉素固体培养基培养载有KLF4序列的细菌,接种环火上灼烧冷却后蘸取质粒菌液,按照“Z”字形划板。半小时后将培养板倒置放在细菌孵箱14小时继续培养。7.按照美国Sigma公司说明书,洗涤、离心、分离、浓缩、裂解、抽提质粒。8.构建LV-KLF4重组慢病毒,利用polybrene提高转染效率,将稀释液再与Enhanced Infection Solution混合均匀,加入进体外培养的细胞中,对KLF4基因进行过表达。荧光显微镜下观察荧光表达情况。放入孵箱继续培养,等荧光稳定后开始用嘌呤霉素筛选。9.使用添加外源性BMP4和Noggin对BMP4信号进行激活或抑制,检测下游分子的表达变化。10.利用特异性单克隆一抗孵育处理后的细胞,用载有荧光标记的二抗结合一抗,再用激光共聚焦细胞免疫荧光检测细胞中各基因的变化。11.使用q RT-PCR和Western blotting方法检测各种处理组基因在m RNA和蛋白质水平的表达变化。结果1.在人类标本中,BE组织高表达BMP4,p-Smad1/5/8 and KLF4。我们用免疫组化SP法,染色证实了在人类BE标本中,BMP4在正常食管组织当中,阳性率9.1%,阳性染色主要集中在细胞质;p-Smad 1/5/8阳性率56.8%,阳性染色也主要集中在细胞核;KLF4阳性率61.4%,阳性染色也主要集中在细胞核。BE组织中,BMP4阳性率94%,阳性染色主要集中在细胞质;p-Smad 1/5/8阳性率96%,阳性染色也主要集中在细胞核;KLF4阳性率96%,阳性染色也主要集中在细胞核。BE组织中BMP4和p-Smad 1/5/8的阳性表达率均显著高于正常食管组织,且差异具有显著性(0.05)。2.在大鼠标本中,BE组织高表达BMP4,p-Smad1/5/8 and KLF4。我们用免疫组化SP法,染色证实了大鼠BE组织中,正常食管组织BMP4阴性表达;p-Smad 1/5/8阴性表达。BE组织中,BMP4阳性表达,阳性染色主要集中在细胞质;p-Smad 1/5/8阳性表达,阳性染色主要集中在细胞核;KLF4阳性表达,阳性染色主要集中在细胞核。BE组织中BMP4、p-Smad 1/5/8和KLF4的阳性表达率均显著高于正常食管组织。3.此外,在体外培养的细胞中,DCA上调了BMP4,KLF4,p-Smad 1/5/8,CDX2,MUC2 and MUC5ac的表达。我们还发现BMP4能够上调KLF4,p-Smad 1/5/8,CDX2,MUC2 and MUC5ac的表达,而Noggin则下调KLF4,p-Smad 1/5/8,CDX2,MUC2 and MUC5ac的表达。另外,经过si RNA-KLF4干扰的细胞,CDX2,MUC2 and MUC5ac的表达下调,此时BMP4不能够再度刺激CDX2,MUC2 and MUC5ac的表达上调。同样,经过慢病毒过表达KLF4的细胞,CDX2,MUC2 and MUC5ac的表达上调,此时Noggin不能够再度引起CDX2,MUC2 and MUC5ac的表达下调。结论1.DCA促进食管上皮细胞BMP4、p-Smad 1/5/8、KLF4以及CDX2,MUC2和MUC5ac的表达。2.外源性BMP4能激活BMP4信号通路,能够上调p-Smad 1/5/8、KLF4以及CDX2,MUC2和MUC5ac的表达。3.KLF4的变化能够影响CDX2,MUC2和MUC5ac的表达。4.DCA所诱导的细胞表型变化是首先通过激活BMP4信号通路,进而引起KLF4上调,最终促进BE分子标志CDX2,MUC2和MUC5ac的表达实现的。
[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.

【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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