赭曲霉毒素A诱导人胃黏膜上皮细胞（GES-1）恶性转化过程中的比较蛋白组学研究

发布时间：2018-01-16 01:04

本文关键词：赭曲霉毒素A诱导人胃黏膜上皮细胞（GES-1）恶性转化过程中的比较蛋白组学研究　出处：《河北医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:赭曲霉毒素A(Ochratoxin A,OTA)是由曲霉菌属和青霉菌属的某些菌株产生的一种真菌毒素,其污染非常普遍,广泛存在于小麦、玉米、豆类等粮食作物以及咖啡、葡萄酒、啤酒、面包等食品中。目前研究已经发现OTA具有肾毒性、肝毒性、神经毒性、免疫毒性、致畸性、致突变性和致癌性等生物学效应。1993年OTA被国际癌症研究中心列为“可能的人类致癌物”。河北省赞皇县是我国胃癌高发区,胃癌年均死亡率超过59/10万。2006年,我们对当地居民粮食中OTA的污染状况进行了现场调查,发现当地居民食用的小麦中OTA的平均含量为2.41μg/kg,明显高于世界卫生组织/粮农组织联合专家委员会(Joint FAO/WHO Expert Committee on Food Additives,JECFA)暂定的每周容许摄入量(100 ng/kg)。提示OTA的高污染可能与胃癌高发区居民胃癌的发生有关。我们前期的研究发现OTA急性暴露可以导致GES-1细胞氧化DNA损伤、细胞周期阻滞以及损伤修复系统的破坏;而长期暴露OTA可增强GES-1细胞的迁移力和侵袭性,诱导细胞发生恶性转化,利用该转化GES-1细胞株成功建立了裸鼠成瘤模型。但是,OTA诱导的GES-1细胞恶性转化的分子机制目前并不清楚。本研究利用恶性转化的GES-1细胞作为研究对象,筛选出OTA诱导GES-1细胞恶性转化过程中的关键分子。我们首先采用双向电泳技术分析GES-1细胞发生恶性转化的过程中蛋白质谱的变化情况,筛选出可能与OTA诱导GES-1细胞恶性转化相关的差异表达蛋白质。然后,在以上研究结果的基础上,利用蛋白印迹、Real-time PCR、免疫组织化学方法,进一步验证双向电泳筛选出的差异蛋白在OTA诱导GES-1细胞恶性转化过程中的作用。寻找OTA长期暴露致人胃黏膜上皮细胞损伤乃至癌变过程中的关键事件,丰富和加深了人们对OTA生物效应的认识,为揭示胃癌高发区居民胃癌发生机制开拓新的研究领域。方法:1细胞培养与处理正常人胃黏膜上皮细胞(GES-1)常规培养在含有10%胎牛血清、100U/ml链霉素、100U/ml青霉素的DMEM培养基中,置于37℃、5%CO2的培养箱中培养,细胞贴壁生长。实验分为OTA处理组和对照组,取对数生长期GES-1细胞,调整细胞浓度为(1~2)×104个/L,接种于培养瓶,细胞培养24h后,给予2.5μmol/L OTA处理72h,一周一次,染毒直至40代后收集细胞进行恶性转化相关指标的检测。前期研究利用裸鼠成瘤实验证实OTA长期处理GES-1细胞后,使GES-1细胞发生了恶性转化。2双向电泳分析技术提取发生恶性转化的GES-1细胞的蛋白样品进行双向电泳分析,通过考马斯亮蓝染色显示凝胶中的蛋白点。计算机比对扫描后,采用Image Master 6.0图像分析软件对选定的凝胶进行分析。然后,将筛选出的差异蛋白点从凝胶中的切出,通过胶内胰蛋白酶解和基质辅助激光解吸电离飞行时间质谱(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry,MALDI-TOF-MS)获得肽质量指纹谱。最后对质谱分析出的蛋白质进行鉴定。3蛋白免疫印迹(Western Blotting)提取细胞总蛋白,用蛋白印迹方法,检测OTA诱导恶性转化的GES-1细胞中双向电泳技术筛选出的差异蛋白(CAPZA1、Annexin A3、GOLPH3L和TRX)的表达情况。4 Real-time PCR提取RNA,反转录成DNA后,利用Real time PCR方法检测OTA处理后CAPZA1、Annexin A3、GOLPH3L和TRX的m RNA水平。5免疫组织化学法将发生恶性转化的GES-1细胞接种裸鼠,于接种后16周将瘤组织取出,并固定,常规石蜡切片,然后利用免疫组织化学方法观察Annexin A3在接种瘤组织中的表达情况。6统计采用SPSS19.0软件进行相关分析与单因素方差分析(analysis of variance,ANOVA),数据用x±s表示,检验以P0.05为差异有显著性意义结果:1筛选与OTA诱导GES-1细胞恶性转化相关的差异表达蛋白在可溶差异蛋白中,恶性转化的GES-1细胞与其平行对照细胞间共有58个差异表达的蛋白质点,其中有14个蛋白点变化最明显,包括表达上调的9个蛋白点和表达下调的5个蛋白点。然后利用MALDI-TOF-MS技术对变化最明显的4个蛋白质点进行鉴定,成功鉴定出4种差异表达蛋白,它们分别是CAPZA1、Annexin A3、GOLPH3L和TRX。其中Annexin A3和GOLPH3L在恶性转化的GES-1细胞中表达均明显上调,而CAPZA1和TRX的表达明显下调。这四种蛋白与细胞骨架构象、细胞内氧化还原反应及信号转导等功能密切相关。2对筛选出的差异表达蛋白进行鉴定蛋白印迹及Real-time PCR检测结果显示经2D电泳筛选出出的4种差异蛋白中Annexin A3和GOLPH3L在恶性转化的GES-1细胞中表达较正常对照组明显上调(P0.05),而CAPZA1和TRX在恶性转化的GES-1细胞中的表达较正常对照组明显下调(P0.05)。其中以Annexin A3变化最为显著。3 Annexin A3在裸鼠接种瘤中的表达情况在裸鼠成瘤标本中Annexin A3的蛋白和m RNA表达水平均明显高于对照组(P0.05)。结果提示Annexin A3蛋白在OTA诱导的GES-1细胞恶性转化中具有重要的作用。结论:1通过蛋白组学技术,筛选出赭曲霉毒素A诱导GES-1细胞恶性转化过程中的差异表达蛋白:Annexin A3、CAPZA1、GOLPH3L和TRX。2 Annexin A3和GOLPH3L的高表达可能参与介导了赭曲霉毒素A诱导的GES-1细胞恶性转化。3 CAPZA1和TRX的低表达可能参与介导了赭曲霉毒素A诱导的GES-1细胞恶性转化。4 OTA长期暴露诱导GES-1细胞发生恶性转化可能是细胞骨架构象改变、细胞内氧化还原反应紊乱以及某些癌基因的激活这三方面因素综合作用的结果。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2

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