【摘要】：一、研究背景與目的DEPTOR作為mTORCl及mTORC2內(nèi)源性抑制因子,在調(diào)控mTOR信號(hào)通路活性起重要作用。研究發(fā)現(xiàn)在多種腫瘤中DEPTOR的表達(dá)被下調(diào),如乳腺癌、前列腺癌、肺癌、慢性髓系白血病,這使得mTOR信號(hào)通路處于過度激活狀態(tài)而促進(jìn)腫瘤生長(zhǎng)。然而在部分多發(fā)性骨髓瘤中DEPTOR卻意外的處于過表達(dá)狀態(tài),DEPTOR的高表達(dá)可抑制mTOR通路的活性,通過負(fù)反饋機(jī)制最終導(dǎo)致Akt的活化,促進(jìn)多發(fā)性骨髓瘤細(xì)胞的存活。目前,已有兩篇文章報(bào)道了DEPTOR與食管癌預(yù)后的關(guān)系。其中一研究結(jié)果顯示:DEPTOR在食管鱗癌患者中的表達(dá)上調(diào),并提示患者較短的生存期。然而,另一研究結(jié)果表明:DEPTOR的表達(dá)在食管鱗癌中下調(diào),并提示較差的預(yù)后。根據(jù)這兩組研究的結(jié)論提示我們,DEPTOR在食管癌中的表達(dá)量及其在預(yù)測(cè)食管鱗狀細(xì)胞癌(ESCC)預(yù)后中的作用,仍未定論,存在一定的爭(zhēng)議。我們本次實(shí)驗(yàn)的目的在于:1、確定DEPTOR在食管癌中的表達(dá)情況,及其對(duì)食管鱗狀細(xì)胞癌預(yù)后的影響。2、DEPTOR對(duì)食管鱗狀細(xì)胞癌化療反應(yīng)的影響。二、研究方法1、在南方醫(yī)院胸外科收集23例食管癌標(biāo)本,將標(biāo)本分為兩部分,一部分進(jìn)行組織固定,包埋、切片及免疫組化染色,觀察DEPTOR蛋白在組織中的表達(dá)部位以及表達(dá)水平;另一部分組織標(biāo)本研磨并提取蛋白,采用免疫印跡的方法檢測(cè)方法檢測(cè)DEPTOR蛋白在組織中的表達(dá)水平。2、于上海芯超公司購買組織芯片2套,共含180例食管癌標(biāo)本。進(jìn)行免疫組化染色,對(duì)DEPTOR的染色結(jié)果進(jìn)行評(píng)分,并做生存期分析。3、選取4種食管癌細(xì)胞系,1種永生化正常食管上皮細(xì)胞系,采用免疫印跡的方式,檢測(cè)該5種細(xì)胞中DEPTOR的表達(dá)水平,并根據(jù)DEPTOR的表達(dá)水平,以永生化正常食管上皮為參考,進(jìn)行分類。4、對(duì)4種食管癌細(xì)胞,培養(yǎng)過程中加入化療藥物,采用CCK-8的檢測(cè)方式,觀察4種食管癌細(xì)胞的存活情況。對(duì)4種食管癌細(xì)胞進(jìn)行干擾和過表達(dá),降低和升高DEPTOR在細(xì)胞中的表達(dá)水平,并加入化療藥物,探究DEPTOR表達(dá)水平的變化,對(duì)細(xì)胞化療敏感性的影響。5、利用免疫印跡方式,探究DEPTOR影響食管癌細(xì)胞化療敏感性的具體分子機(jī)制。三、研究結(jié)果我們的研究結(jié)果顯示DEPTOR在腫瘤組織中的表達(dá)顯著增加,并且在早期ESCC患者中提示較高的生存率。體外研究顯示,可根據(jù)正常食管鱗狀上皮細(xì)胞系HET-1A中的DEPTOR表達(dá)量,將ESCC細(xì)胞系分為相對(duì)高和低DEPTOR表達(dá)的亞群。在我們的研究中,不同水平的DEPTOR表達(dá)影響著食管癌細(xì)胞系對(duì)化療藥物的反應(yīng)。在相對(duì)低表達(dá)的細(xì)胞系中,DEPTOR通過降低Akt活性增加化療敏感性。在相對(duì)高表達(dá)的細(xì)胞系中,DEPTOR通過在下調(diào)核糖體蛋白S6激酶(S6K)后負(fù)反饋激活I(lǐng)RS1-P13K-Akt-Survivin通路,從而增加細(xì)胞的化療耐藥。
[Abstract]:Background and objective DEPTOR, as an intrinsic inhibitor of mTORCl and mTORC2, plays an important role in regulating the activity of mTOR signaling pathway. It has been found that the expression of DEPTOR is down-regulated in a variety of tumors, such as breast cancer, prostate cancer, lung cancer and chronic myeloid leukemia, which makes the mTOR signaling pathway overactivated and promotes tumor growth. However, DEPTOR is unexpectedly overexpressed in some multiple myeloma. The high expression of DEPTOR can inhibit the activity of mTOR pathway, and eventually lead to the activation of Akt through negative feedback mechanism, and promote the survival of multiple myeloma cells. At present, two articles have reported the relationship between DEPTOR and prognosis of esophageal carcinoma. One of the results showed that the expression of DEPTOR was up-regulated in patients with esophageal squamous cell carcinoma and suggested a shorter survival time. However, another study showed that the expression of DEPTOR was down-regulated in esophageal squamous cell carcinoma and suggested poor prognosis. According to the conclusions of these two groups, the expression of DEPTOR in esophageal carcinoma and its role in predicting the prognosis of esophageal squamous cell carcinoma (ESCC) are still uncertain and controversial. The purpose of our experiment was to determine the expression of DEPTOR in esophageal carcinoma and its effect on the prognosis of esophageal squamous cell carcinoma. 2, the effect of DEPTOR on chemotherapy response of esophageal squamous cell carcinoma. 2. Methods 1. 23 specimens of esophageal carcinoma were collected in thoracic surgery of Southern Hospital. The specimens were divided into two parts, one of which was fixed, embedded, sectioned and stained by immunohistochemistry, and the expression site and expression level of DEPTOR protein in the tissue were observed. The other part of the tissue specimens were ground and extracted, and the expression of DEPTOR protein in the tissues was detected by immunoblotting. 2. Two sets of tissue chips were purchased in Shanghai Xinchao Company, including a total of 180 esophageal cancer specimens. The results of DEPTOR staining were evaluated by immunohistochemical staining, and the survival time was analyzed. 3. Four kinds of esophageal cancer cell lines and one immortalized normal esophageal epithelial cell line were selected to detect the expression of DEPTOR in the five kinds of cells by immunoblotting. According to the expression level of DEPTOR, 4 kinds of esophageal cancer cells were classified with reference to immortalized normal esophageal epithelial cells. Chemotherapeutic drugs were added in the culture process and CCK-8 was used to observe the survival of four kinds of esophageal cancer cells. Four kinds of esophageal cancer cells were interfered with and overexpressed, the expression level of DEPTOR in cells was decreased and increased, and chemotherapeutic drugs were added to explore the change of DEPTOR expression level and the effect on the chemosensitivity of esophageal cancer cells. 5. The specific molecular mechanism of DEPTOR affecting the chemosensitivity of esophageal cancer cells was explored by immunoblotting. Third, our results showed a significant increase in the expression of DEPTOR in tumor tissues, and suggested a higher survival rate in patients with early ESCC. In vitro studies showed that ESCC cell lines could be divided into relatively high and low DEPTOR subpopulations according to the expression of DEPTOR in normal esophageal scaly epithelial cell line HET-1A. In our study, different levels of DEPTOR expression affected the response of esophageal cancer cell lines to chemotherapeutic drugs. In relatively low expression cell lines, DEPTOR increased chemotherapy sensitivity by reducing Akt activity. In the cell lines with relatively high expression, DEPTOR activated the IRS1-P13K-Akt-Survivin pathway by negative feedback after down-regulating ribosomal protein S6 kinase (S6K), thus increasing the chemoresistance of the cells.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 María José Domper Arnal;ángel Ferrández Arenas;ángel Lanas Arbeloa;;Esophageal cancer: Risk factors,screening and endoscopic treatment in Western and Eastern countries[J];World Journal of Gastroenterology;2015年26期

2 Yuwei Zhang;;Epidemiology of esophageal cancer[J];World Journal of Gastroenterology;2013年34期

3 ;Epidemiologic differences in esophageal cancer between Asian and Western populations[J];癌癥;2012年06期

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本文編號(hào)：2502949