本文關(guān)鍵詞：赭曲霉毒素A誘導(dǎo)人胃黏膜上皮細(xì)胞（GES-1）惡性轉(zhuǎn)化過(guò)程中的比較蛋白組學(xué)研究　出處：《河北醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 赭曲霉毒素 人永生化胃黏膜上皮細(xì)胞 惡性轉(zhuǎn)化 蛋白組學(xué) Annexin A3

【摘要】：目的:赭曲霉毒素A(Ochratoxin A,OTA)是由曲霉菌屬和青霉菌屬的某些菌株產(chǎn)生的一種真菌毒素,其污染非常普遍,廣泛存在于小麥、玉米、豆類等糧食作物以及咖啡、葡萄酒、啤酒、面包等食品中。目前研究已經(jīng)發(fā)現(xiàn)OTA具有腎毒性、肝毒性、神經(jīng)毒性、免疫毒性、致畸性、致突變性和致癌性等生物學(xué)效應(yīng)。1993年OTA被國(guó)際癌癥研究中心列為“可能的人類致癌物”。河北省贊皇縣是我國(guó)胃癌高發(fā)區(qū),胃癌年均死亡率超過(guò)59/10萬(wàn)。2006年,我們對(duì)當(dāng)?shù)鼐用窦Z食中OTA的污染狀況進(jìn)行了現(xiàn)場(chǎng)調(diào)查,發(fā)現(xiàn)當(dāng)?shù)鼐用袷秤玫男←溨蠴TA的平均含量為2.41μg/kg,明顯高于世界衛(wèi)生組織/糧農(nóng)組織聯(lián)合專家委員會(huì)(Joint FAO/WHO Expert Committee on Food Additives,JECFA)暫定的每周容許攝入量(100 ng/kg)。提示OTA的高污染可能與胃癌高發(fā)區(qū)居民胃癌的發(fā)生有關(guān)。我們前期的研究發(fā)現(xiàn)OTA急性暴露可以導(dǎo)致GES-1細(xì)胞氧化DNA損傷、細(xì)胞周期阻滯以及損傷修復(fù)系統(tǒng)的破壞;而長(zhǎng)期暴露OTA可增強(qiáng)GES-1細(xì)胞的遷移力和侵襲性,誘導(dǎo)細(xì)胞發(fā)生惡性轉(zhuǎn)化,利用該轉(zhuǎn)化GES-1細(xì)胞株成功建立了裸鼠成瘤模型。但是,OTA誘導(dǎo)的GES-1細(xì)胞惡性轉(zhuǎn)化的分子機(jī)制目前并不清楚。本研究利用惡性轉(zhuǎn)化的GES-1細(xì)胞作為研究對(duì)象,篩選出OTA誘導(dǎo)GES-1細(xì)胞惡性轉(zhuǎn)化過(guò)程中的關(guān)鍵分子。我們首先采用雙向電泳技術(shù)分析GES-1細(xì)胞發(fā)生惡性轉(zhuǎn)化的過(guò)程中蛋白質(zhì)譜的變化情況,篩選出可能與OTA誘導(dǎo)GES-1細(xì)胞惡性轉(zhuǎn)化相關(guān)的差異表達(dá)蛋白質(zhì)。然后,在以上研究結(jié)果的基礎(chǔ)上,利用蛋白印跡、Real-time PCR、免疫組織化學(xué)方法,進(jìn)一步驗(yàn)證雙向電泳篩選出的差異蛋白在OTA誘導(dǎo)GES-1細(xì)胞惡性轉(zhuǎn)化過(guò)程中的作用。尋找OTA長(zhǎng)期暴露致人胃黏膜上皮細(xì)胞損傷乃至癌變過(guò)程中的關(guān)鍵事件,豐富和加深了人們對(duì)OTA生物效應(yīng)的認(rèn)識(shí),為揭示胃癌高發(fā)區(qū)居民胃癌發(fā)生機(jī)制開(kāi)拓新的研究領(lǐng)域。方法:1細(xì)胞培養(yǎng)與處理正常人胃黏膜上皮細(xì)胞(GES-1)常規(guī)培養(yǎng)在含有10%胎牛血清、100U/ml鏈霉素、100U/ml青霉素的DMEM培養(yǎng)基中,置于37℃、5%CO2的培養(yǎng)箱中培養(yǎng),細(xì)胞貼壁生長(zhǎng)。實(shí)驗(yàn)分為OTA處理組和對(duì)照組,取對(duì)數(shù)生長(zhǎng)期GES-1細(xì)胞,調(diào)整細(xì)胞濃度為(1~2)×104個(gè)/L,接種于培養(yǎng)瓶,細(xì)胞培養(yǎng)24h后,給予2.5μmol/L OTA處理72h,一周一次,染毒直至40代后收集細(xì)胞進(jìn)行惡性轉(zhuǎn)化相關(guān)指標(biāo)的檢測(cè)。前期研究利用裸鼠成瘤實(shí)驗(yàn)證實(shí)OTA長(zhǎng)期處理GES-1細(xì)胞后,使GES-1細(xì)胞發(fā)生了惡性轉(zhuǎn)化。2雙向電泳分析技術(shù)提取發(fā)生惡性轉(zhuǎn)化的GES-1細(xì)胞的蛋白樣品進(jìn)行雙向電泳分析,通過(guò)考馬斯亮藍(lán)染色顯示凝膠中的蛋白點(diǎn)。計(jì)算機(jī)比對(duì)掃描后,采用Image Master 6.0圖像分析軟件對(duì)選定的凝膠進(jìn)行分析。然后,將篩選出的差異蛋白點(diǎn)從凝膠中的切出,通過(guò)膠內(nèi)胰蛋白酶解和基質(zhì)輔助激光解吸電離飛行時(shí)間質(zhì)譜(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry,MALDI-TOF-MS)獲得肽質(zhì)量指紋譜。最后對(duì)質(zhì)譜分析出的蛋白質(zhì)進(jìn)行鑒定。3蛋白免疫印跡(Western Blotting)提取細(xì)胞總蛋白,用蛋白印跡方法,檢測(cè)OTA誘導(dǎo)惡性轉(zhuǎn)化的GES-1細(xì)胞中雙向電泳技術(shù)篩選出的差異蛋白(CAPZA1、Annexin A3、GOLPH3L和TRX)的表達(dá)情況。4 Real-time PCR提取RNA,反轉(zhuǎn)錄成DNA后,利用Real time PCR方法檢測(cè)OTA處理后CAPZA1、Annexin A3、GOLPH3L和TRX的m RNA水平。5免疫組織化學(xué)法將發(fā)生惡性轉(zhuǎn)化的GES-1細(xì)胞接種裸鼠,于接種后16周將瘤組織取出,并固定,常規(guī)石蠟切片,然后利用免疫組織化學(xué)方法觀察Annexin A3在接種瘤組織中的表達(dá)情況。6統(tǒng)計(jì)采用SPSS19.0軟件進(jìn)行相關(guān)分析與單因素方差分析(analysis of variance,ANOVA),數(shù)據(jù)用x±s表示,檢驗(yàn)以P0.05為差異有顯著性意義結(jié)果:1篩選與OTA誘導(dǎo)GES-1細(xì)胞惡性轉(zhuǎn)化相關(guān)的差異表達(dá)蛋白在可溶差異蛋白中,惡性轉(zhuǎn)化的GES-1細(xì)胞與其平行對(duì)照細(xì)胞間共有58個(gè)差異表達(dá)的蛋白質(zhì)點(diǎn),其中有14個(gè)蛋白點(diǎn)變化最明顯,包括表達(dá)上調(diào)的9個(gè)蛋白點(diǎn)和表達(dá)下調(diào)的5個(gè)蛋白點(diǎn)。然后利用MALDI-TOF-MS技術(shù)對(duì)變化最明顯的4個(gè)蛋白質(zhì)點(diǎn)進(jìn)行鑒定,成功鑒定出4種差異表達(dá)蛋白,它們分別是CAPZA1、Annexin A3、GOLPH3L和TRX。其中Annexin A3和GOLPH3L在惡性轉(zhuǎn)化的GES-1細(xì)胞中表達(dá)均明顯上調(diào),而CAPZA1和TRX的表達(dá)明顯下調(diào)。這四種蛋白與細(xì)胞骨架構(gòu)象、細(xì)胞內(nèi)氧化還原反應(yīng)及信號(hào)轉(zhuǎn)導(dǎo)等功能密切相關(guān)。2對(duì)篩選出的差異表達(dá)蛋白進(jìn)行鑒定蛋白印跡及Real-time PCR檢測(cè)結(jié)果顯示經(jīng)2D電泳篩選出出的4種差異蛋白中Annexin A3和GOLPH3L在惡性轉(zhuǎn)化的GES-1細(xì)胞中表達(dá)較正常對(duì)照組明顯上調(diào)(P0.05),而CAPZA1和TRX在惡性轉(zhuǎn)化的GES-1細(xì)胞中的表達(dá)較正常對(duì)照組明顯下調(diào)(P0.05)。其中以Annexin A3變化最為顯著。3 Annexin A3在裸鼠接種瘤中的表達(dá)情況在裸鼠成瘤標(biāo)本中Annexin A3的蛋白和m RNA表達(dá)水平均明顯高于對(duì)照組(P0.05)。結(jié)果提示Annexin A3蛋白在OTA誘導(dǎo)的GES-1細(xì)胞惡性轉(zhuǎn)化中具有重要的作用。結(jié)論:1通過(guò)蛋白組學(xué)技術(shù),篩選出赭曲霉毒素A誘導(dǎo)GES-1細(xì)胞惡性轉(zhuǎn)化過(guò)程中的差異表達(dá)蛋白:Annexin A3、CAPZA1、GOLPH3L和TRX。2 Annexin A3和GOLPH3L的高表達(dá)可能參與介導(dǎo)了赭曲霉毒素A誘導(dǎo)的GES-1細(xì)胞惡性轉(zhuǎn)化。3 CAPZA1和TRX的低表達(dá)可能參與介導(dǎo)了赭曲霉毒素A誘導(dǎo)的GES-1細(xì)胞惡性轉(zhuǎn)化。4 OTA長(zhǎng)期暴露誘導(dǎo)GES-1細(xì)胞發(fā)生惡性轉(zhuǎn)化可能是細(xì)胞骨架構(gòu)象改變、細(xì)胞內(nèi)氧化還原反應(yīng)紊亂以及某些癌基因的激活這三方面因素綜合作用的結(jié)果。
[Abstract]:Objective: ochratoxin A (Ochratoxin A OTA) is a kind of mycotoxin produced by some strains of Aspergillus and Penicillium. The pollution is very common, widely exist in wheat, corn, beans and other food crops, coffee, Wine, beer, bread and other foods. The current study has found OTA has kidney toxicity, liver toxicity, neurotoxicity, immune toxicity, teratogenicity, mutagenicity and carcinogenicity and other biological effects of.1993 OTA by the international cancer research center as a "probable human carcinogen". Hebei County of Zanhuang province is a high incidence of gastric cancer in China, gastric cancer annual mortality rate more than 59/10 million.2006 year, we local residents in grain OTA pollution status of the scene investigation, found that the average content of local residents to eat wheat in OTA was 2.41 g/kg, significantly higher than the WHO / FAO Joint Expert Committee (Joint FAO/WHO E Xpert Committee on Food Additives, JECFA) of the provisional tolerable weekly intake (100 ng/kg). The high pollution and residents in the high incidence area of gastric cancer gastric cancer suggests that OTA related to the occurrence of OTA. Our previous studies found that acute exposure can cause oxidative DNA damage in GES-1 cells, cell cycle arrest and repair system damage and long-term exposure; OTA can enhance the migration and invasion of GES-1 cells, induce cell malignant transformation, the transformation of GES-1 cell line was successfully established in nude mice model. However, the molecular mechanism of GES-1 cell malignant transformation induced by OTA is not clear. This study uses the malignant transformation of GES-1 cells as the research object, selected the key molecules the process of malignant transformation induced by OTA in GES-1 cells. We used two-dimensional electrophoresis analysis of proteins of GES-1 cell malignant transformation in the The situation, screening and OTA may induce GES-1 cell malignant transformation related protein. Then, based on the above results, using Western blot, Real-time PCR, immunohistochemistry, further validation of the process of malignant transformation between 2D electrophoresis of protein in GES-1 cells induced by OTA in the role of key events. Looking for long-term OTA exposure induced human gastric epithelial cell damage and carcinogenesis, enrich and deepen the understanding of the biological effects of OTA, in order to reveal the residents in the high incidence area of gastric cancer gastric cancer mechanism to explore new areas of research. Methods: 1 cell culture and treatment of normal human gastric mucosal epithelial cells (GES-1) cultured in the presence of 10% fetal bovine serum, 100U/ml streptomycin, penicillin 100U/ml DMEM medium at 37 DEG C, the cultivation box 5%CO2, adherent cells were divided into OTA treatment group. And the control group, GES-1 cells, the cell concentration was adjusted to 104 * /L (1~2), inoculated in culture bottles, cell culture 24h, given 2.5 mol/L OTA 72h, once a week, until the 40 generation cells were collected after the detection of relevant indicators of malignant transformation of previous research by exposure. The nude mice confirmed that OTA long-term treatment after GES-1 cell malignant transformation.2 electrophoresis extraction of malignant transformation of GES-1 cell protein samples analysis technique of 2-DE gel showed that GES-1 cells, the protein by Coomassie blue staining. The computer scanning, using Image Master 6 image analysis software analysis of the selected gel. Then, the selected proteins from the gel were cut out, through the gel trypsin digestion and matrix assisted laser desorption ionization time-of-flight mass spectrometry (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry, MALDI-TOF-MS) for peptide mass fingerprinting. The mass spectrometry proteins were identified by Western blot of.3 (Western Blotting) and total protein were extracted, using Western blotting method, the difference of two-dimensional electrophoresis screened protein detection of OTA induced malignant transformation of GES-1 cells (CAPZA1. Annexin A3, GOLPH3L and TRX) on the expression of.4 Real-time PCR RNA extraction, reverse transcription DNA, detection of OTA using Real time PCR method Annexin A3, m CAPZA1, RNA.5 and TRX GOLPH3L immunohistochemical method to malignant transformation of GES-1 cells in nude mice 16 weeks after inoculation the tumor tissue removed, and fixed, paraffin, then use immunohistochemical method to observe the Annexin A3 inoculation in tumor tissues. The expression of.6 system using SPSS19.0 software Analysis of correlation analysis and single factor variance (analysis of, variance, ANOVA), expressed by X + s test data, P0.05 had significant difference results in malignant transformation of the differentially expressed proteins in the soluble proteins in GES-1 cells induced by 1 screening and OTA, malignant transformation of GES-1 cells and total parallel control 58 differentially expressed protein spots between cells, of which 14 protein spots including the most obvious changes, and the expression of 9 protein spots were up-regulated and 5 were down regulated protein spots. Then the use of MALDI-TOF-MS technology to the most obvious change of the 4 egg white particles were identified and successfully identified 4 differential protein expression, they are CAPZA1 Annexin, A3, GOLPH3L and TRX. were significantly up-regulated in Annexin A3 and GOLPH3L in the malignant transformation of GES-1 cells, and the expression of CAPZA1 and TRX were down regulated. These four proteins and the cytoskeleton conformation, The intracellular redox reaction and signal transduction function is closely related to.2 of the differentially expressed proteins were identified by Western blotting and Real-time PCR results showed that 4 kinds of Annexin proteins in A3 and GOLPH3L by 2D electrophoresis screening out in malignant transformation of GES-1 cells was significantly increased compared with normal control group (P0.05), and the expression of CAPZA1 and TRX in malignant transformation in GES-1 cells compared with normal control group was significantly reduced (P0.05). The Annexin A3 change is the most significant.3 Annexin expression of A3 in nude mice tumor the expression level were significantly higher than the control group of Annexin in the samples of A3 protein and m RNA in nude mice (P0.05) A3. The results suggest that Annexin protein plays an important role in the malignant transformation of GES-1 cells induced by OTA. Conclusion: 1 by proteomics technology, screening of malignant transformation of GES-1 cells induced by ochratoxin A The differentially expressed proteins in the process of Annexin: A3, CAPZA1, GOLPH3L and TRX.2 Annexin high expression of A3 and GOLPH3L may be involved in mediating the ochratoxin A GES-1 cells induced by low expression of.3 CAPZA1 and TRX of the malignant transformation may mediate ochratoxin A induced GES-1 cell malignant transformation of.4 OTA exposure induction of GES-1 cell malignant transformation may alter cytoskeletal conformation, intracellular redox reaction results in activation of some oncogenes and disorder of the comprehensive effect of these three factors.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.2

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