【摘要】：研究背景： 我國是食管癌高發(fā)地區(qū)，其中超過90%的食管癌患者的病理類型為鱗狀細(xì)胞癌。食管鱗癌的病因?qū)W及發(fā)病學(xué)機(jī)制迄今尚不明確。研究表明，食管鱗癌的發(fā)生和發(fā)展是一個多基因參與、多階段誘變的過程，很多異常表達(dá)的基因在食管鱗狀細(xì)胞的惡性轉(zhuǎn)化過程中起了關(guān)鍵作用。從分子生物學(xué)水平研究食管鱗狀細(xì)胞癌的發(fā)生發(fā)展，對食管鱗狀細(xì)胞癌的診斷、預(yù)防和治療具有重要的意義�；蛐酒�(lián)合生物信息學(xué)技術(shù)，從整體上動態(tài)地、定量地觀察腫瘤過程中關(guān)鍵基因的改變，能全面了解腫瘤發(fā)病機(jī)制，為腫瘤的預(yù)防及診治提供新的思路。 羥基前列腺素脫氫酶（hydroxyprostaglandin dehydrogenase，HPGD）是前列腺素降解的關(guān)鍵酶，同時對環(huán)氧合酶（cyclooxygenase, COX-2）具有生理性拮抗作用。近來研究顯示，HPGD的表達(dá)缺失或減少可能與一些惡性腫瘤，如前列腺癌、結(jié)腸癌、胃癌、肺癌、乳腺癌、甲狀腺癌、肝癌等的發(fā)生發(fā)展密切相關(guān)。 研究目的: 1、通過基因芯片和深入的生物信息學(xué)分析技術(shù)，全面分析食管鱗癌事件進(jìn)程中基因及相關(guān)通路等改變，以期找到該過程中起關(guān)鍵作用的基因及通路。 2、初步明確候選關(guān)鍵基因-HPGD對食管鱗癌增殖和侵襲等生物學(xué)作用，初步探討其在致食管鱗癌事件中的可能機(jī)制。 材料與方法: 1、收集外科手術(shù)切除的8對食管鱗癌和配對正常食管粘膜新鮮組織標(biāo)本，應(yīng)用基因芯片（Affymetric GeneChip Human Genome U133plus2.0Array）以及生物信息學(xué)分析尋找兩組之間的差異表達(dá)基因（differentially expressed genes，DEGs），對篩查出的差異表達(dá)基因分別進(jìn)行基因本體（Gene ontology，GO）功能富集度分析和Pathway功能分析，以及構(gòu)建基因共表達(dá)網(wǎng)絡(luò)。 2、對候選差異基因HPGD進(jìn)一步功能驗(yàn)證和初步機(jī)制探討。首先在線應(yīng)用Oncomine、GEOdataset共享的芯片數(shù)據(jù)，熒光定量PCR及Western blot方法檢測HPGD在食管癌組織及正常食管組織的表達(dá)情況。對60對福爾馬林固定、石蠟包埋的食管鱗癌組織及配對癌旁正常組織標(biāo)本作為研究對象。采用免疫組織化學(xué)的方法檢測HPGD及COX-2蛋白在組織標(biāo)本中的表達(dá)情況，并進(jìn)行臨床病理參數(shù)的相關(guān)性分析。以6株食管鱗癌細(xì)胞（TE1、TE2、TE8、KYSE150、KYSE410、EC9706）和一株正常食管上皮細(xì)胞株（Het-1A），采用熒光定量PCR方法和Western blot方法分別檢測HPGD、COX-2的mRNA和蛋白的表達(dá)情況。篩選出相對高表達(dá)（TE1）和低表達(dá)（TE2）的食管癌細(xì)胞株。構(gòu)建HPGD功能區(qū)全長表達(dá)載體以及靶向HPGD的shRNA載體，分別轉(zhuǎn)染至相對高表達(dá)的細(xì)胞株和低表達(dá)的細(xì)胞株。分別利用熒光定量PCR和Western blot的方法檢測HPGD及COX-2在轉(zhuǎn)染前后mRNA及蛋白的表達(dá)變化。應(yīng)用四甲基偶氮唑鹽微量酶反應(yīng)比色法(MTT)、BD bioCoatTMMatrigel侵襲小室等檢測HPGD過表達(dá)或沉默后食管鱗癌細(xì)胞增殖和侵襲能力及相關(guān)基因的變化。應(yīng)用甲基化特異性PCR（MSP）及亞硫酸鹽鹽測序方法（BSP）檢測ESCC組織、癌旁組織及細(xì)胞株中HPGD啟動子區(qū)域甲基化情況。應(yīng)用去甲基化制劑5-氮雜脫氧胞（5-aza-dC）處理TE2細(xì)胞株后，應(yīng)用RT-PCR方法檢測mRNA表達(dá)情況。 結(jié)果： 1、8對食管鱗癌組織及配對食管正常粘膜組織制備基因芯片后，繪制差異表達(dá)基因譜，共檢出1208個差異表達(dá)基因，其中529個表達(dá)上調(diào)、679個表達(dá)下調(diào)。GO分析發(fā)現(xiàn)，上調(diào)差異基因顯著性功能過程包括細(xì)胞外基質(zhì)組成及分解、細(xì)胞周期、細(xì)胞粘附、細(xì)胞增殖等。下調(diào)基因主要參與小分子代謝、角化細(xì)胞分化、花生四烯酸代謝、鈣離子依賴性細(xì)胞間粘附、上皮細(xì)胞增殖等過程。Pathway分析顯示，上調(diào)差異基因參與的顯著性信號轉(zhuǎn)導(dǎo)通路是ECM受體相互作用通路、局部粘附通路、PI3K-Akt通路等，下調(diào)差異基因參與的顯著性信號通路有代謝通路、花生四烯酸代謝通路、依賴細(xì)胞色素P450的藥物代謝通路、細(xì)胞粘附分子（CAMs）通路。在構(gòu)建的基因共表達(dá)網(wǎng)絡(luò)中，我們篩選共表達(dá)地位變化大的差異基因，對其中之一HPGD擬進(jìn)一步研究。 2、經(jīng)驗(yàn)證HPGD在食管鱗癌組織中表達(dá)下調(diào)，與基因芯片相一致。免疫組化顯示，HPGD在41例食管鱗癌組織呈陰性表達(dá)，COX-2在46例食管鱗癌組織中呈陽性表達(dá)，相關(guān)性分析顯示兩者在食管鱗癌表達(dá)呈負(fù)相關(guān)。HPGD蛋白的陰性表達(dá)、COX-2蛋白的陽性表達(dá)與食管鱗癌患者的淋巴結(jié)轉(zhuǎn)移及是否累及外膜呈正相關(guān)(P＜0.05)，而與患者的性別、年齡、腫瘤細(xì)胞分化程度、腫瘤大小等無顯著相關(guān)性（P＞0.05）。熒光定量PCR及Western blot發(fā)現(xiàn)HPGD在正常食管上皮細(xì)胞株Het-1A中表達(dá)，，在其他四個細(xì)胞株表達(dá)具有不同程度的表達(dá)下調(diào)。我們選擇表達(dá)較低的ESCC細(xì)胞株TE2和表達(dá)較高的ESCC細(xì)胞株TE1。構(gòu)建過表達(dá)及沉默HPGD的載體，分別轉(zhuǎn)染至TE2和TE1細(xì)胞株。MTT顯示，過表達(dá)HPGD后可抑制TE2細(xì)胞增殖，沉默HPGD表達(dá)后可促進(jìn)TE1細(xì)胞增殖（P＜0.01）。Matrigel Transwell遷移和侵襲實(shí)驗(yàn)顯示，過表達(dá)HPGD后可抑制TE2細(xì)胞遷移與侵襲，沉默HPGD表達(dá)后可促進(jìn)TE1細(xì)胞遷移與侵襲（P＜0.01）。MSP、BSP檢測Het-1A、TE8中HPGD啟動子呈完全非甲基化，TE2細(xì)胞株呈完全甲基化，其他三株細(xì)胞株呈部分甲基化。TE2、EC9706細(xì)胞經(jīng)去甲基化藥物5-aza-dC處理后顯示HPGD mRNA表達(dá)增加。 結(jié)論： 1、經(jīng)基因芯片聯(lián)合生物信息學(xué)技術(shù)分析，篩選出HPGD是關(guān)鍵的下調(diào)差異表達(dá)基因之一，具有多種顯著靶向性功能，參與腫瘤關(guān)鍵信號通路。 2、HPGD在食管鱗癌組織中存在表達(dá)缺失或下調(diào)，影響食管鱗癌細(xì)胞的增殖和侵襲能力。HPGD與COX-2在食管鱗癌中表達(dá)模式相反。HPGD食管鱗癌中發(fā)揮抑癌作用，啟動子區(qū)DNA甲基化可能在食管鱗癌發(fā)生發(fā)展中起一定作用。
[Abstract]:Study Background: In the region of high incidence of esophageal cancer, more than 90% of the patients with esophageal cancer are of squamous cell type. The etiology and pathogenesis of squamous cell carcinoma of the esophagus are not yet unknown. The research shows that the occurrence and development of esophageal squamous cell carcinoma is a multi-gene and multi-stage mutation, and many abnormal expression genes play a key role in the malignant transformation of esophageal squamous cell. To study the development of esophageal squamous cell carcinoma from the level of molecular biology, it is of great importance to the diagnosis, prevention and treatment of esophageal squamous cell carcinoma. The gene chip is combined with the bioinformatics technology to dynamically and quantitatively observe the change of the key gene in the tumor from the whole, can comprehensively understand the pathogenesis of the tumor, and provide a new thinking for the prevention and the diagnosis and treatment of the tumor. The hydroxyprostaglandin dehydrogenase (HPGD) is a key enzyme for the degradation of prostaglandins, and has a physiological function on the cyclooxygenase (COX-2). Recent studies have shown that the deletion or reduction of the expression of HPGD may be associated with a number of malignant tumors, such as prostate cancer, colon cancer, gastric cancer, lung cancer, breast cancer, thyroid cancer, liver cancer, etc. Cutting-related. Objective: To study the changes of gene and related pathway in the process of esophageal squamous cell carcinoma by gene chip and in-depth bioinformatics analysis, in order to find a key role in this process. The biological effects of the candidate key gene-HPGD on the proliferation and invasion of esophageal squamous cell carcinoma were discussed. the incidence of cancer in the cancer event may Mechanisms, materials and methods:1,8 pairs of esophageal squamous cell carcinoma and paired normal esophageal mucosa fresh tissue specimens were collected, and the differential expression genes were found between the two groups using the gene chip (Affymetric GeneChip Human Genome U133plus2.0 Array) and the bioinformatics analysis. The gene ontology (GO) function enrichment analysis and the pathway function were carried out on the screened differentially expressed genes, respectively. And constructing a gene co-expression network. D. First, on-line application of Oncomine, GEOdataset shared chip data, fluorescence quantitative PCR and Western blot to detect the HPGD in the esophagus. The expression of cancerous tissue and normal esophageal tissue.60 cases of esophageal squamous cell carcinoma with formalin-fixed and paraffin-embedded tissues and its preparation The expression of HPGD and COX-2 protein in tissue specimens was detected by immunohistochemistry. The clinical and pathological parameters were analyzed. Six esophageal squamous cell lines (TE1, TE2, TE8, KYSE150, KYSE410, EC9706) and a normal esophageal epithelial cell line (Het-1A) were used. Expression of mRNA and protein of-2. Relative high expression (TE1) was selected. The invention discloses an esophageal cancer cell line with low expression (TE2), a full-length expression vector of the HPGD functional area and an shRNA vector targeting the HPGD are constructed, High-expression cell lines and low-expression cell lines, HPGD and COX-2 were detected by fluorescence quantitative PCR and Western blot respectively. The expression of mRNA and protein was detected before and after dyeing. The expression of HPGD and the expression of HPGD and the expression of HPGD were detected by the method of tetramethyl azo-salt microzyme reaction (MTT), BD bioCoatTMMatrigel invasion cell, etc. Proliferation and invasion ability and the change of related genes. The methylation specific PCR (MSP) and the sulfite salt sequencing method (BSP) were used to detect ESCC, adjacent tissues and cell lines. In that case of region methylation of the HPGD promoter, RT-PCR was used to treat the TE2 cell strain by demethylation preparation 5-aza-deoxy-cell (5-aza-dC). PCR Methods The expression of mRNA was detected. Results: After the gene chip was prepared by 1,8 pairs of esophageal squamous cell carcinoma and the normal mucosa of the paired esophagus, the differentially expressed genes were drawn, and 1208 differentially expressed genes were detected. Of these,529 were up-regulated and 679 down-regulated. GO analysis found that the significant sexual function of the up-regulated differential gene included extracellular matrix composition. and the down-regulation genes are mainly involved in small molecule metabolism, keratinocyte differentiation, arachidonic acid metabolism, calcium separation, Pathway analysis showed that the significant signal transduction pathway of the upregulation of the differential gene was the interaction pathway of the ECM, the local adhesion pathway, the PI3K-Akt pathway, and the significance of the down-regulation of the differential gene. The signal pathway has a metabolic pathway, a arachidonic acid metabolic pathway, a drug dependent on cytochrome P450, Metabolic pathway, cell adhesion molecule (CAMs) pathway. In the constructed gene co-expression network, we screened the difference in co-expression status heterogene, one of that HPGD is to be further study.2, validated HPGD The expression of COX-2 in esophageal squamous cell carcinoma (OSCC) was negative in 41 cases of esophageal squamous cell carcinoma, and the expression of COX-2 was positive in 46 cases of esophageal squamous cell carcinoma. The negative expression of the HPGD protein, the positive expression of the COX-2 protein and the lymph node metastasis of the patients with esophageal squamous cell carcinoma and the involvement of the adventitia were positively correlated (P <0.05). The expression of HPGD in the normal esophageal epithelial cell line Het-1A was found by fluorescence quantitative PCR and Western blot. The expression of the other four cell lines has a different degree of expression down-regulation. We choose to express a lower ESCC fine Cell strain TE2 and the high-expression ESCC cell line TE1. The expression and silencing of HP were constructed. The vector of GD was transfected into TE2 and TE1 cell lines respectively. MTT showed that after the expression of the HPGD, the proliferation of TE2 cells and the silence of the HPGD were inhibited. The proliferation of TE1 cells can be promoted after expression (P <0.01). Matrigel Transwell migration and invasion experiments show that after the expression of the HPGD, the migration and invasion of the TE2 cells can be inhibited, and the expression of the HPGD is silent. It can promote the migration and invasion of TE1 cells (P <0.01). The MSP and BSP test Het-1A, and the HPGD promoter in TE8 is completely non-methylated, and TE2 cells The strain was completely methylated and the other three cell lines were partially methylated. TE2 and EC9706 cells were demethylated 5-az. a-d The results showed that the expression of HPGD mRNA was increased after C treatment. One of the most significant targeted sexual functions involved in the tumor-critical signaling pathway. Lack of expression or down-regulation in the presence of expression, affecting the proliferation of esophageal squamous cell carcinoma cells and The expression pattern of the HPGD and the COX-2 in the esophageal squamous cell carcinoma is opposite to that of the expression pattern of the COX-2 in the squamous cell carcinoma of the esophagus.
【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.1

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相關(guān)期刊論文 前3條

1 ;Effects of cyclooxygenase-2 on human esophageal squamous cell carcinoma[J];World Journal of Gastroenterology;2011年41期

2 ;15-PGDH is reduced and induces apoptosis and cell cycle arrest in gastric carcinoma[J];World Journal of Gastroenterology;2012年10期

3 Wanqing Chen;Rongshou Zheng;Hongmei Zeng;Siwei Zhang;Jie He;;Annual report on status of cancer in China, 2011[J];Chinese Journal of Cancer Research;2015年01期

本文編號：2483765