甲醇中毒视网膜功能结构和蛋白质组学研究

发布时间：2018-01-20 03:44

本文关键词： 甲醇视网膜视网膜电图蛋白质组学信号转导　出处：《复旦大学》2012年博士论文　论文类型：学位论文

【摘要】：甲醇是重要的化工原料和有机溶剂,是工业用酒精的主要成分之一,由于其价格低廉、来源广泛,且具有微弱的乙醇香味,时常被一些不法分子用于制造假酒,引起群体性中毒事件并造成恶劣的社会影响；在生活和生产中,通过皮肤接触或是呼吸道吸入致甲醇急慢性中毒的案例也时有发生。急性中毒多因误服甲醇代替乙醇作为饮料所致,由于多数患者不会意识到饮用假酒这一事实,临床诊断和治疗往往不够及时和准确,甚至于漏诊,以至于造成极其严重的后果。中毒者以视力障碍和神经系统症状较为突出,严重者甚至于死亡,并且即使经过抢救等及时治疗,视力障碍也通常难以完全恢复。因甲醇中毒死亡或致残的,多会酿成严重的刑事案件或民事纠纷。中毒后幸存者往往需要鉴定损伤程度和伤残等级的评定,有时也会涉及中毒与不良后果之间因果关系的鉴定,其中又常涉及视觉功能检验,这就需要我们对甲醇中毒对视网膜的毒性作用和损害机制有全面、深入的了解。作为甲醇毒性的一个重要靶器官,视网膜的宏观功能、微观结构、分子组成等方面在甲醇的毒性作用下都会发生一定改变。视网膜电图作为视网膜功能的客观检测方法之一,在临床上被广泛作为视网膜疾病与视网膜损伤的诊断和治疗依据。由于视网膜电图各波形可以反应视网膜不同细胞的生物电活动,所以视网膜电图不仅可用于评估甲醇对视网膜造成的宏观功能损害,而且可以在细胞层面反映视网膜不同细胞的受损情况。近年来蛋白质组学逐渐在毒理学研究方面崭露头角,作为高通量的蛋白质研究方法,其不仅可用于筛选生物学标志物,也可为毒理学作用机制的研究提供新的方法。本课题在原有研究基础和研究方法上,将研究甲醇对视网膜功能、形态以及蛋白质分子的毒性作用,并探讨甲醇对视网膜的毒性作用机制。主要的研究内容为以下6个部分：第一部分甲醇中毒视网膜功能改变研究为研究不同甲醇剂量和不同毒性作用时间下的视网膜功能,建立了甲醇中毒的实验大鼠模型(FR大鼠),并依据给毒剂量和毒性作用时间分为中毒剂量3天组,中毒剂量7天组和低剂量3天组、低剂量7天组。依据ISCEV(International Society for Clinical Electrophysiology of Vision)规定方法,分别在暗视和明视状态下进行大鼠视网膜电图的检测,结合统计学方法研究甲醇对视网膜视杆细胞反应、最大混合反应和视锥细胞反应的影响。研究发现：甲醇毒性致视网膜功能呈现不同程度下降,中毒剂量甲醇对明视、暗视系统损害都较大,暗视系统损害较重呈现进行性下降,而明视系统损害进展缓慢。较低剂量的甲醇会造成以暗视系统为主的一过性损害。第二部分视网膜与肝肾等代谢相关组织病理形态学改变研究为了研究视网膜在甲醇毒性作用下的组织学改变,以及组织结构随时间改变的特征,我们特取视网膜组织进行光学显微镜和透射电子显微镜观察其显微结构和超微结构变化。同时取部分肝肾组织进行组织学比较研究,以研究视网膜对甲醇毒性的敏感性。组织经过固定、脱水、包埋、切片、染色等流程,在光学显微镜下视网膜呈现细胞水肿和组织疏松,以外层视网膜为甚。透射电子显微镜下,发现线粒体水肿、空泡、嵴断裂等表现。但不同剂量和不同毒性作用时间下的视网膜结构改变特点有所不同：中毒剂量的甲醇会造成光感受器和内核层细胞组织形态的改变,并且随中毒时间延长组织学改变有加重趋势；光感受器的线粒体损害早于内核层线粒体损害的发生；低剂量甲醇对视网膜组织结构的毒性作用是可逆的；视网膜较其他器官对甲醇更为敏感。第三部分甲醇血浓度的检测与分析为研究甲醇的代谢情况,利用顶空气相色谱法检测各组实验动物血中的甲醇浓度。顶空气相色谱法是一种可稳定准确检测血甲醇的定性定量方法。将各组实验动物引颈处死后,取心血及时保存于EDTA抗凝管中,将1uL的血样与500uL叔丁醇混合,直接进入顶空进样器,每个样本经检测器FID1A和FID2B检查两次。研究发现甲醇的保留时间为1.134min；中毒剂量的甲醇在给药7天后仍可被检测到,说明甲醇在体内代谢缓慢,存在一定蓄积性。第四部分甲醇中毒视网膜蛋白质组学研究为研究甲醇中毒后视网膜蛋白质组学的变化,提取临床中毒症状和视网膜功能障碍最严重的中毒剂量7天组和对正常对照组的视网膜蛋白质并定量,经过双向凝胶电泳分离、凝胶染色和图像采集分析,认定灰度相差3倍以上为的蛋白质为差异蛋白,并胶上酶解差异蛋白,经过MALDI-TOF-TOF MS质谱鉴定,成功筛选出24个差异蛋白质,其中上调的蛋白包括14个,如aldehyde dehydrogenase, tropomyosin alpha-1chain, myosin light chain MLC1, myosin regulatory light chain2, actin gamma1propeptide-like, myosin light chain MLC3, alpha-crystalline A chain (2spots), alpha-crystalline B chain(3spots), beta-crystalline A2, beta-crystalline B2, and beta-crystalline B3下调的蛋白包括10个,如heat shock protein60, actin cytoplasmic1, glyceraldehyde-3-phosphate dehydrogenase,tubulin beta-2A chain, recoverin, synuclein Beta, Chain A Calmodulin, ATP synthase (H+transporting, mitochondrial F1complex, alpha subunit), ATP synthase subunit alpha, zero beta-globin. a晶状体蛋白B经western blot验证确系明显上调。差异蛋白种类和功能涉及到细胞骨架相关蛋白、与代谢及线粒体功能相关蛋白、视网膜特有结构和功能蛋白。从毒性反应分析,甲醇对视网膜蛋白质造成的影响一方面在于甲醇及其代谢物对视网膜组织蛋白的直接毒性作用,包括对细胞骨架相关蛋白和对线粒体功能相关蛋白的损害；另一方面的影响在于引发视网膜自身对毒性作用所做出的保护性反应,如晶体蛋白的分子伴侣功能。第五部分差异性蛋白的亚细胞定位为研究差异性蛋白质的亚细胞定位,采用LOCtree在线服务软件,输入24个差异性蛋白质的氨基酸序列,得到每个蛋白质的亚细胞定位信息。其中位于细胞胞浆的蛋白质最多,高达75%,其次是线粒体16.67%,位于细胞外以及与DNA结合的蛋白质均为4.17%。亚细胞定位信息提示除线粒体功能障碍这一经典作用途径外,可能存在线粒体外的其他作用机制,或是多条途径相互影响的情况。第六部分构建甲醇中毒视网膜差异性蛋白质基因间的信号转导网络为研究差异性蛋白间潜在的信号关联和中毒机制,基于KEGG数据库,在数据库中搜索满足筛选条件基因的上游和下游基因,之后根据基因与其上、下游相互作用关系画出基因间连接线。分析发现,ATP5A1(ATP合成酶基因)是中介能力最强的基因；与ATP5A1相互作用的基因数目最多为高达40个,所以ATP5A1在整个Signal-Net中处于一个关键节点的位置；位于ATP5A1的下游基因包括细胞骨架蛋白、甲醛脱氢酶以及晶体蛋白。再次提示线粒体是重要的中介部位,甲醇可能除了直接影响线粒体代谢而导致视网膜损害外,还对位于线粒体中的ATP合成酶的改变进而影响到其下游的细胞骨架蛋白、甲醛脱氢酶、以及晶体蛋白等的调节和功能。小结通过对视网膜功能和结构的影响,发现了不同剂量和不同中毒时间的甲醇对视网膜功能和形态结构的影响不同,甲醇对内核层和光感受器细胞均有损害,光感受器细胞线粒体损害早于内核层线粒体的损害,视杆细胞较视锥细胞对甲醇更为敏感；运用顶空气相色谱法,确证了甲醇在体内代谢缓慢情况,并发现甲醇浓度的降低不会终止视网膜功能和结构的进一步恶化；通过对甲醇中毒后视网膜蛋白质组学的研究,首次筛选出了甲醇对视网膜毒性作用的候选生物学标志物；通过对差异性蛋白的亚细胞定位研究和构建差异蛋白质的信号转导网络,提示线粒体处于甲醇毒性机制的中心位置外,存在线粒体外的毒性作用机制。
[Abstract]:Methanol is an important chemical raw material and organic solvent , it is one of the main components of industrial alcohol . Because of its low cost , wide source , and weak alcohol flavor , it is often difficult to fully recover from acute poisoning due to the fact that alcohol is used instead of ethanol as beverage . As an important target organ of the toxicity of methanol , the macroscopic function , microstructure and molecular composition of the retina can change under the toxic action of methanol . As one of the objective detection methods of retinal function , the electroretinogram is widely used as the basis for diagnosis and treatment of retinal diseases and retinal damage . In recent years , proteomic research has gradually emerged in toxicology research . As a high - throughput protein research method , it can be used not only to screen biological markers , but also to provide new methods for the research of toxicological mechanism . Study on the changes of retinal function in the first part of methanol poisoning In order to study the retinal function in rats with different methanol dose and different toxic action time , the rats model of methanol poisoning ( FR rats ) was established , and the effects of methanol on the response , maximal mixing reaction and optic cone cell response were studied . Study on pathological changes of metabolism - related tissues in the second part of retina and liver and kidney In order to study the histological changes of retina in the toxic action of methanol and the change of tissue structure with time , we studied the microstructure and ultrastructure changes of retina in the light microscope and transmission electron microscope . The Detection and Analysis of the Blood Concentration of the Third Part of Methanol In order to study the metabolism of methanol , the methanol concentration in blood of each group was determined by headspace gas chromatography . Study on the Proteomics of Retinal Proteomics in the Fourth Part of Methanol Poisoning In order to study the changes of retinal protein group after methanol poisoning , the most serious poisoning dose of 7 days and the amount of retinal protein in normal control group were extracted , and 24 differentially expressed proteins were identified by two - way gel electrophoresis separation , gel staining and image acquisition . ATP synthase ( H + ATPase , mitochondrial F1complex , alpha subunit ) , ATP synthase subunit alpha , zero beta - globin . a lens protein B was confirmed by western blot . The effects of methanol and its metabolites on retinal tissue protein are the direct toxic effects of methanol and its metabolites on retinal tissue proteins , including the damage to cytoskeletal - related proteins and proteins associated with mitochondrial function , and on the other hand , the protective effects of the retina itself on the toxic effect , such as the molecular - mate function of crystallin . Subcellular Localization of the Fifth Differential Protein In order to study the sub - cell localization of differential protein , the amino acid sequence of 24 different proteins was input by using the LOCtree on - line service software , and the sub - cell location information of each protein was obtained . The protein located in the cytoplasm of the cells was up to 75 % , followed by the mitochondrial 16.67 % , the protein located outside the cell and the DNA binding protein was 4 . 17 % . The sub - cell location information suggested that there might be other mechanisms of action outside the mitochondria , or multiple ways of interacting with each other , except for the classical pathway of mitochondrial dysfunction . Part 6 : Construction of signal transduction network between protein gene of retinal difference in methanol poisoning It is found that ATP5A1 ( ATP synthase gene ) is the most intermediate gene in the whole Signal - Net . small knot The effects of methanol on retinal function and morphological structure were found by the effects of methanol on retinal function and morphological structure .

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：D919.4;R779.13

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