本文選題：食管鱗癌　切入點(diǎn)：WASH　出處：《鄭州大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景和目的食管癌是一種常見(jiàn)的消化道惡性腫瘤。近些年來(lái),食管癌的發(fā)病率明顯上升,因此針對(duì)食管癌發(fā)病機(jī)理的研究以及發(fā)現(xiàn)食管癌的治療靶點(diǎn)迫在眉睫。在我國(guó),食管癌患者的5年生存率不足20%,食管鱗癌的發(fā)病率占食管癌的90%,因此在本文主要是針對(duì)食管鱗癌的研究。越來(lái)越多的報(bào)道顯示,細(xì)胞骨架的意義已經(jīng)超出了只做為細(xì)胞形態(tài)結(jié)構(gòu)的概念。近年研究發(fā)現(xiàn),細(xì)胞的骨架結(jié)構(gòu)及功能的改變也是導(dǎo)致腫瘤發(fā)生和發(fā)展的一個(gè)必不可少的重要因素。且在很多研究中表明,腫瘤細(xì)胞的惡性表型通常與肌動(dòng)蛋白相關(guān)蛋白的異常表達(dá)相關(guān)。在本研究中,我們所關(guān)注的細(xì)胞骨架調(diào)節(jié)蛋白WASH,又叫WASHC1,是WASP(Wiskott-Aldrich syndrome Protein)家族中一種重要的細(xì)胞骨架調(diào)節(jié)相關(guān)蛋白。在腫瘤中,有關(guān)WASH細(xì)胞骨架調(diào)節(jié)蛋白參與腫瘤發(fā)生發(fā)展尚鮮見(jiàn)報(bào)道。作為一種重要的細(xì)胞骨架調(diào)節(jié)蛋白,WASH是否影響腫瘤細(xì)胞的命運(yùn),能否影響腫瘤細(xì)胞的進(jìn)展和轉(zhuǎn)移等等,這些問(wèn)題都值得進(jìn)一步的探討和研究。本課題的主要任務(wù)是一方面鑒定WASH在食管鱗癌的發(fā)生發(fā)展中所起的重要作用;另一方面找到WASH影響食管鱗癌進(jìn)展的機(jī)制,進(jìn)一步結(jié)合臨床探究其意義。方法收集84例食管鱗癌患者的癌組織及配對(duì)的癌旁組織,采用熒光定量PCR檢測(cè)WASH在基因水平的表達(dá),并分析WASH的表達(dá)與病人病理參數(shù)的相關(guān)性,以及其與病人生存相關(guān)性;利用免疫組化實(shí)驗(yàn)檢測(cè)WASH在不同分期食管鱗癌腫瘤組織中的表達(dá)情況;在3株食管鱗癌細(xì)胞系中,檢測(cè)WASH在食管鱗癌腫瘤干細(xì)胞和非干細(xì)胞的表達(dá)情況;利用基因沉默技術(shù)沉默WASH之后,探究食管鱗癌細(xì)胞的成球情況,通過(guò)熒光定量和westernblot檢測(cè)部分干性基因的表達(dá);通過(guò)瞬時(shí)轉(zhuǎn)染技術(shù)過(guò)表達(dá)WASH之后,檢測(cè)食管癌細(xì)胞系的成球情況及干性基因的表達(dá)情況;在利用sh RNA技術(shù)沉默WASH之后,多因子試劑盒、熒光定量PCR及ELISA檢測(cè)細(xì)胞因子的表達(dá)差異,并探究其上下游機(jī)制;動(dòng)物實(shí)驗(yàn)探究WASH在裸鼠體內(nèi)的生長(zhǎng)情況,探究WASH的表達(dá)對(duì)腫瘤進(jìn)展的影響;為了探究WASH的上游機(jī)制,利用網(wǎng)站預(yù)測(cè)及雙熒光報(bào)告等實(shí)驗(yàn)探究與其靶向的micro RNA,并在45例病人中檢測(cè)此micro RNA的表達(dá)情況,檢測(cè)與臨床參數(shù)相關(guān)性,探究micro RNA與WASH的相關(guān)性,分析mircro RNA與病人預(yù)后相關(guān)性。結(jié)果1.熒光定量PCR結(jié)果顯示:WASH在癌組織比癌旁組織高表達(dá),差異有明顯統(tǒng)計(jì)學(xué)意義。WASH的表達(dá)與病理分期有明顯相關(guān)性。2.免疫組化結(jié)果顯示:WASH在癌組織比癌旁組織高表達(dá),差異有明顯統(tǒng)計(jì)學(xué)意義。WASH的表達(dá)與病理分期及分化有明顯相關(guān)性。且WASH與病人預(yù)后成負(fù)相關(guān)。3.成球?qū)嶒?yàn)結(jié)果顯示:沉默WASH之后,食管鱗癌細(xì)胞系成球減少,差異具有統(tǒng)計(jì)學(xué)差異。且干性基因的表達(dá)也降低。瞬時(shí)過(guò)表達(dá)WASH之后,食管鱗癌細(xì)胞系成球增多,干性基因表達(dá)增加。4.多因子檢測(cè)實(shí)驗(yàn)結(jié)果顯示:沉默WASH后,細(xì)胞因子CXCL8的表達(dá)明顯降低。差異具有統(tǒng)計(jì)學(xué)差異。5.動(dòng)物實(shí)驗(yàn)結(jié)果顯示:皮下注射穩(wěn)定敲低WASH的細(xì)胞系,相對(duì)于對(duì)照組腫瘤體積及質(zhì)量顯著減少。在小鼠的腫瘤組織中,相對(duì)于對(duì)照組,敲低WASH后,CXCL8的表達(dá)顯著降低。6.通過(guò)targetscan,mi Rbase等預(yù)測(cè)網(wǎng)站及利用雙熒光報(bào)告實(shí)驗(yàn)檢測(cè)出mi R-637靶向結(jié)合WASH的3’-UTR。并且在瞬時(shí)轉(zhuǎn)染mi R-637之后,WASH的表達(dá)顯著降低。并且通過(guò)構(gòu)建mi R-637的穩(wěn)定過(guò)表達(dá)系統(tǒng),發(fā)現(xiàn)WASH的表達(dá)顯著降低。在45例食管鱗癌臨床病人中,mi R-637在癌旁組織比癌組織高表達(dá),差異有明顯統(tǒng)計(jì)學(xué)意義。在臨床病人中,mi R-637表達(dá)與WASH的表達(dá)有顯著負(fù)相關(guān)。結(jié)論1.WASH在食管鱗癌癌組織中是高表達(dá)的。WASH促進(jìn)了食管鱗癌細(xì)胞的干性,促進(jìn)了食管鱗癌的發(fā)生發(fā)展。2.在食管鱗癌中,低表達(dá)的mi R-637導(dǎo)致WASH的上調(diào),進(jìn)而WASH通過(guò)上調(diào)CXCL8促進(jìn)食管鱗癌細(xì)胞的干性,促進(jìn)食管鱗癌的進(jìn)展。
[Abstract]:Background and objective: esophageal cancer is a common malignant tumor of digestive tract. In recent years, the incidence of esophageal cancer increased significantly, so the study on the pathogenesis of esophageal cancer and the discovery of therapeutic targets of esophageal cancer is imminent. In China, the 5 year survival rate of less than 20% of patients with esophageal cancer, the incidence rate of esophageal squamous cell carcinoma for esophageal cancer 90%, so in this paper is mainly study on esophageal squamous cell carcinoma. More and more reports indicate that the cytoskeleton has significance beyond just as the concept of cell morphology. Recent studies have found that skeleton structure and function of cells can also lead to the change of tumor occurrence and development of an indispensable factor. And that in many cases, the abnormal expression of malignant phenotype of tumor cells is associated with actin protein. In this study, we focus our attention on the regulation of cytoskeletal protein WASH , also known as WASHC1, WASP (Wiskott-Aldrich syndrome Protein) is a kind of important cytoskeletal associated protein family. In tumors, the WASH cytoskeletal protein involved in tumor development was rarely reported. As an important cytoskeletal protein, the influence of WASH on tumor cell fate, and can progress the impact of the transfer of tumor cells and so on, these issues are worthy of further study and research. The main task of this project is on the one hand in the identification of WASH plays an important role in the development of esophageal squamous cell carcinoma; on the other hand, to find the mechanism of WASH on the progression of esophageal squamous cell carcinoma, further explore the combined with clinical significance. Methods patients with esophageal squamous cell carcinoma 84 cases of cancer tissues and paired adjacent tissues, the expression by fluorescence quantitative PCR detection of WASH at the gene level, and analyze the expression and pathological parameters of the patients with WASH Correlation between patient survival and its correlation; expression by immunohistochemical assay of WASH in different stages of esophageal squamous cell carcinoma tissues; in 3 strains of esophageal squamous cell carcinoma cell lines, detection of WASH stem cells and the expression of non stem cells in esophageal squamous cell carcinoma tumor; after silencing WASH using gene silencing technology, a ball study of esophageal carcinoma cells by fluorescence quantitative detection expression of stemness genes and Westernblot; overexpression of WASH by transfection technique, ball and dry into gene detection of esophageal carcinoma cell line expression; after using WASH sh RNA silencing technology, multi factor kit, differential expression and fluorescence quantitative PCR ELISA detection of cytokines, and to explore its downstream mechanism; animal experiment research of WASH growth in nude mice, the expression of WASH on influence on tumor progression in order to exploration; The upstream mechanism of the WASH, using the site prediction and double fluorescence report and experiment inquiry targeted micro RNA, and the expression of micro RNA was detected in 45 patients, correlation detection and clinical parameters of micro and RNA, to explore the relevance of WASH, RNA and Mircro analysis of prognosis correlation. Results 1. fluorescent quantitative PCR the results showed that WASH high expression in cancer tissue than in adjacent tissues, the difference has statistical significance of.WASH expression and pathological stage there was significant correlation between.2. immunohistochemistry results showed that: the expression of WASH in cancer tissue than in adjacent tissue, there are significant differences in the expression of.WASH and pathological stage and differentiation and have significant correlation. The prognosis of WASH patients with a negative correlation of.3. into the ball experiment results showed that silencing of WASH, esophageal squamous cell carcinoma cell lines into the ball is reduced, a statistically significant difference. The expression of genes is reduced. And the dry instant After overexpression of WASH in esophageal squamous cell carcinoma cell line, into the ball more, dry gene expression increased.4. results in multiple factor detection experiments showed that silencing of WASH and cytokine CXCL8 expression significantly decreased. A statistically significant difference.5. animal experiment results showed that subcutaneous injection of stable knockdown of WASH cell line, tumor volume and relative to the control group the quality was significantly reduced in mice. The tumor tissues, compared with the control group, knockdown of WASH, the expression of CXCL8 was significantly reduced by.6. targetscan, MI Rbase and predict the site with dual luciferase reporter experiments detected mi R-637 target WASH 3 '-UTR. and R-637 MI after transfection, the expression of WASH significantly reduced. And through the construction of MI R-637 stable expression system, expression of WASH decreased significantly. In 45 patients of esophageal squamous cell carcinoma, MI R-637 in cancer tissues than in cancer tissues with high expression And there was a significant difference. In clinical patients, MI expression of R-637 and WASH have significant negative correlation. Conclusion 1.WASH in esophageal squamous cell carcinoma is high expression of.WASH promoted esophageal squamous cell dry, promote the occurrence and development of esophageal squamous cell carcinoma.2. in esophageal squamous carcinoma, low expression of MI R-637 leads to the upregulation of WASH and WASH, through the upregulation of CXCL8 promote esophageal squamous cell carcinoma and promoting the progress of the dry, esophageal squamous cell carcinoma.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.1

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相關(guān)期刊論文 前2條

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本文編號(hào)：1619519