【作者】 王双双；

【导师】 王慧君；

【作者基本信息】 南方医科大学 ， 法医学， 2010， 硕士

【摘要】 研究背景甲基苯丙胺(Methamphetamine,METH)属于苯丙胺类兴奋剂(amphetamine-type stimulants, ATS),是我国规定管制的精神药品。苯丙胺药物强烈的兴奋作用以及相对易于制作的特性,使它们刚应用于临床不久就开始被滥用。冰毒虽然问世较晚,但它见效快、药效维持时间长,其最大的特点是第一次使用便可能会上瘾,因此,被称为“毒品之王”。近年国际和国内大中城市的滥用情况十分严峻。1996年联合国禁毒署国际兴奋剂的专家会议上,一致认为ATS将逐步取代本世纪流行的鸦片、海洛因、大麻、可卡因等常用毒品,成为21世纪全球范围滥用最为广泛的新型毒品之一。联合国毒品和犯罪问题办事处(UNODC)对全球各个国家和地区调查显示,在全球有3000多万人滥用ATS,滥用人群更趋低龄化、女性化。我国非法使用ATS的问题也日益严重,临床上因ATS的滥用而导致各种生理、心理及精神障碍者屡见报道。此药滥用不仅给个人生理及心理带来极大痛苦,而且给家庭及社会带来沉重负担。METH具有药物依赖性(主要是精神依赖性)、中枢神经兴奋和拟交感效应等药理、毒理学特性。研究证明,METH对中枢神经系统、心、肝、肾和骨骼肌等组织器官均有毒性作用。近年来研究发现,METH相关的心脏疾病可能是导致滥用者猝死的重要原因之一。已有研究表明,METH可刺激周围神经系统释放大量的儿茶酚胺类物质,引起血管收缩、血管痉挛、心动过速和血压增高。这可能跟METH毒理学特性和拟交感效应的药理学作用有关。本文希望通过METH的急慢性毒性对心脏损伤的研究,观察METH对大鼠心肌的毒性损害及心脏功能的影响。而在METH的神经毒性方面,目前国内外的研究结果尚不足以阐明METH的神经毒性机制。在本教研室前一阶段的研究中,检测到一种新型的NO生成调节酶二甲基精氨酸二甲基氨基水解酶1 (Nω,Nω-diMAyl-L-arginine diMAylaminohydrolase1,DDAH1)在纹状体和皮质内高表达。DDAH主要参与不对称二甲精氨酸(asymmetric diMAylated L-arginine, ADMA)的分解代谢。而ADMA是内源性的一氧化氮合酶(nitric oxide synthase, NOS)抑制剂,对三种亚型的NOS均有抑制作用。DDAH通过调节组织内ADMA的降解率,从而调节组织中NOS的活性和一氧化氮(nitric oxide, NO)的合成。NOS的活性和NO的合成增加可导致大量的活性氮产生,引起蛋白质的亚硝酰化、硝基化、氧化损伤等。其中蛋白质酪氨酸硝化被认为是普遍存在的蛋白质翻译后修饰,能引起蛋白功能的获得或丧失。蛋白质是细胞功能的最终执行者,细胞关键蛋白质的损伤,有可能导致细胞相关功能的丧失直至细胞死亡。经研究,在肌萎缩性侧索硬化、帕金森病、阿尔兹海默病等多种神经退行性疾病中,多种关键蛋白质酪氨酸残基的硝化及硝化水平的增加是这些疾病病理过程的重要环节。在前面两项研究的基础上,我们尝试采用血清蛋白质组学方法对METH的整体机体毒性进行探索性研究。血清蛋白质组学是建立在正常蛋白质表达图谱的基础上,研究选定的目标人群血清中表达的全部蛋白质,寻找其差异蛋白质点,鉴定疾病相关蛋白质,进而研究其结构和功能的一种新方法,将为研究重大疾病病理生理学机制、早期诊断的特异性标志物、药物作用靶点等开辟新的途径。METH急性中毒可能会导致滥用者体内蛋白质数量和质量的改变,可能在分子水平揭示METH成瘾机制。血清蛋白质可能含有生物标记物,可区分机体生理或病理状态,也许可作为甲基苯丙胺机体损伤的一个预示、筛查和诊断。目的尝试运用行为学、病理学、神经化学等方法,多方位、全面了解METH对机体的毒性损伤及损伤机制,并初步筛选可预示机体损伤的血清标志物。方法1、METH急慢性中毒大鼠的心脏毒性观察Wistar大鼠制成METH急性及慢性中毒动物模型,各自设立对照组。对照组注射等量生理盐水,余处理同实验组。用药后观察大鼠的行为变化并进行评分；监测其心率及血压变化。造模结束后用10%的水合氯醛腹腔注射麻醉,腹主动脉促凝管采集血液标本并分离血清,采用双抗夹心酶联免疫吸附法(ELISA)测定血清BNP和Tn-T。迅速打开胸腔摘取心脏,大体观察称重、HE染色观察有无组织病理学改变。2、甲基苯丙胺急性中毒鼠大脑纹状体组织的神经毒性作用Wistar大鼠制成METH急性中毒动物模型及其对照组,造模结束后断头处死。快速断头取脑,冰上分离双侧纹状体,硝酸还原酶法测定纹状体NO含量,化学比色法测定各类NOS的活性,免疫印迹法测定纹状体内硝基酪氨酸水平即蛋白质硝化程度。3、METH急性中毒大鼠血清内差异蛋白质的表达Wistar大鼠制成METH急性中毒动物模型及其对照组。造模结束后用10%的水合氯醛(3ml／kg)腹腔注射麻醉,内眦静脉促凝管采集血液标本2 ml,以3000r/min离心10 min,取上清置-80℃冰箱保存备用,双向凝胶电泳结合质谱检测方法检测血清内差异蛋白的表达。结果1.慢性METH中毒大鼠注射METH后有明显的直立探索、攀爬、鼻嗅等行为增多,行为学评分与对照组相比有显著性差异(F=602.887,P<0.001)；心率和平均动脉压显著高于对照组(F=2723.644,P<0.001；F=413.556,P<0.001)；大鼠心肌大体观察未见明显异常；HE染色切片可见心肌细胞排列紊乱,可见粗颗粒样变、嗜酸性变、细胞空泡性变、局灶性肌纤维溶解、断裂以及纤维性变；慢性METH组大鼠血清中Tn-T和BNP含量均显著升高,差异有统计学意义(r=10.188,P<0.001；t=10.716,P<0.001)。2.急性METH中毒大鼠注射METH后亦有行为明显增多,主要为：直立探索、攀爬、鼻嗅等动作,行为学评分与对照组相比有显著性差异(F=349.057,P<0.001)；心率和平均动脉压显著高于对照组(F=1051.599,P<0.001；F=373.974,P<0.001)；心肌大体观察未见明显异常；HE染色切片可见局灶性心肌纤维断裂,肌溶灶形成并可见部分区域心肌收缩带坏死；血清中Tn-T含量明显升高,差异有显著性意义(t=9.566,P<0.001),血清BNP的含量无显著性变化(t=1.407,P=0.177)3.METH急性中毒鼠大脑纹状体区NO含量和NOS活性显著性升高(t=23.067,P<0.001；t=13.419,P<0.001)；蛋白质酪氨酸硝基化水平显著升局。4.双向电泳检测出急性METH中毒大鼠血清内有差异蛋白点16个,质谱检测有意义的有10个,在血清内表达上调的蛋白质为：Glial fibrillary acidic protein, haptoglobin (Hp), Carcinoembryonic antigen-related cell adhesion molecule 3 precursor等3个。表达下调的蛋白质为GTP-binding protein Rab-3D, Testis-specific serine kinase substrate, Ras-related protein Rab-3A, Sodium/hydrogen exchanger 3, Advanced glycosylation end product -specific receptor precursor, Alpha-1-inhibitor 3 precursor等6个。结论1.METH短期大剂量和长期应用均可对大鼠心肌造成明显的毒性损伤：可引起刻板行为增多,心率和血压升高,心脏切片可观察到明显的心肌细胞损伤,造成心肌损伤标志物Tn-T的升高。急性METH中毒因用药时间较短,并未引起心脏功能的损害,而慢性METH中毒则可引起BNP含量的明显升高,说明已经对心脏功能造成影响。2.METH急性中毒可引起脑纹状体区NO含量及NOS活性的增高,进而引起蛋白质硝化水平的升高。蛋白质硝化可引起蛋白质功能的受损或丧失。说明METH导致功能蛋白的酪氨酸硝基化进而导致这些功能蛋白功能受损或丧失可能是METH中枢神经毒性的一个重要原因。3.从急性METH中毒大鼠的血清内中共获得有差异蛋白点16个,其中经过质谱检测,有意义的有10个。这些蛋白主要参与细胞骨架蛋白重构、信号转导／物质转运／大分子代谢、酶活性失调等,而它们的受损可能是METH对机体毒性损伤的主要作用机制。血清内蛋白变化可作为预示METH毒性的一个指标,并为METH机体毒性提供一定的机制说明。更多还原

【Abstract】 BACKGROUNDMethamphetamine (MA) is known as methylamphetamine, which belongs to the amphetamine-type stimulants (ATS). This drug is one of psychotropic substances which are controlled by our country. Because it has strong excitement actions and is easy to produce, it was abused rapidly after being applied to clinic. Though methamphetamine is a new drug, it was widely abused for its instant and long time effect. The most important feature of it is that people will be addicted for the first use. So it is called the Drug Lord. Recently the drug abuse gets more and more serious in the word and many major cities in China. Experts agreed that this drug will spread to all round the world and will become to one of the main drugs in 21st century. The global investigation of United Nations Office on Drugs and Crime (UNODC) showed that by 2004, there were 30 million people abuse in the world. The drugger’s age become lower ages and the sex to trend feminize, even to rich. Recently the drugs of usurpation get more and more serious. We can know a lot of druggers from the magazines. It brings the disasters not only to the person in the physiology and the mind but also to the family and the society.The drug was found to have much psychic dependence, stimulant action on the central nervous system and sympathomimetic action. Some researches show that METH has severe injury to central nervous system, heart, kidney and skeletal muscle. And METH related heart diseases may be one of most important causes which induce druggers’ sudden death. MTHE can cause vasoconstriction, vasospasm, tachycardia and high blood pressure through stimulating the peripheral nervous system to release huge amounts of catecholamines. This paper will carry out studies aimed at the toxicity of METH on heart and heart function.The METH-induced neurotoxicity is a complicated process that via many ways and mechanisms. The definite mechanism has not been known, so there is no effective method to prevent it. Our form discussing group found that the increased DDAH1 (Nω,Nω-diMAyl-L-arginine diMAylaminohydrolase, DDAH) in cerebral striatum and cortex which is a new regulatory enzyme to help the synthesis of NO. DDAH takes part in the catabolism of ADMA(asymmetric diMAylated L-arginine, ADMA), which is an endogenic inhibitor of NOS and can inhibit three subtypes of NOS. So DDAH regulates the activity of NOS and the synthesis of NO via adjusting the degradation of ADMA. The increase of the activity of NOS and the synthesis of NO can produce a great amount of Reactive Nitrogen Species (RNS), which will cause nitrosation, nitration and oxidation of proteins. And tyrosine nitration of proteins is an important posttranslational modification, leading to the gain or lose of protein functions. Protein is the final executant of life functions and the damage of the key proteins will bring about the lose of cell functions and the death of cells. Protein tyrosine nitration can be detected through identify 3-nitrotyrosine with special antibody. Many researches show that the nitration of key proteins plays an important role in many neurodegenerative diseases such as amyotrophic lateral sclerosis, Parkinson disease and Alzheimer’s disease.On the basis of the previous two studies, we tried to utilize serum proteomicson to explore the METH toxicity on the whole organism. Serum proteomics is a new method, which is based on the normal protein expression profiles, to study the expression of serum total protein of the target population, look for differences between the proteins, and identify disease-related proteins, and then study the structures and functions of the proteins. It will help to study the pathophysiology mechanism of major diseases, find specific markers for early diagnosis, and research drug targets. Acute METH toxicity may lead to the changes in quantity and quality of body’s protein. Which may reveal the mechanism of METH addiction at the molecular level. Besides, serum proteins may contain biological markers to distinguish physiological or pathological state of the body.OBJECTIVETry to study the toxicity of METH on rats and mechanism by histopathological, behavioral and neurochemical methods. On the bases of these results, proteomic method was used to screen serum proteins which may be biological markers to show the body damage associated with METH toxicity.METHODS1. Observation on heart toxicity of acute and chronic METH abuseWistar rats, weighting from 180g to 220g (the Laboratory Animal Center of Southern Medical University, China) were made two kinds of METH toxicity models-the acute one and chronic one. And the control group was set up respectively. The control group was injected with the identical volume of saline. The behavior changes, heart rate and blood pressure were observed. The rats were anaesthetized and killed and trunk blood was collected in serum tubes for the determination of serum Tn-T and BNP. Rapidly, the hearts were removed, weighed and gross observed. The changes of heart were observed by HE staining.2. The neurotoxicity in striatum of acute METH exposed ratsWistar rats were made acute METH toxicity models.And set up the control group which was injected with the identical volume of saline. The rats were anaesthetized and killed. Rapidly, the striatum were removed on ice. To detect the concentration of NO in striatum homogenate by nitrate reductase method; To detect the activity of NOS with colorimetry; To detect the contents of 3-nitrotyrosine with western blot.3. Proteomic profiling of serum proteins associated with METH-induced toxicityProteins of serum were separated by two-dimensional gel electrophoresis. The different expressed proteins were identified by tandem mass spectrum and data base search.RESULTS1. In acute METH toxicity models, the times of stereotyped behavior were increased obviously, mainly including quest, shinny, nasil and so on; The grades of behavior were increased obviously (P<0.001); The heart rate and mean arterial pressure(MAP) increased significantly than the control groups; The gross observation of hearts were normal; local myofibril fracture and dissolution, as well as contraction band necrosis were found in HE staining section; The content of Tn-T in serum rose obviously (P<0.05), but that of BNP increased without statistical significance.2. In chronic METH toxicity models, the times of stereotyped behavior were increased obviously, mainly including quest, shinny, nasil and so on; The grades of behavior were increased obviously (P<0.001); The heart rate and MAP increased significantly than the control groups; The gross observation of hearts were normal; acidophilic degeneration, vacuolar degeneration, local myofibril fracture and dissolution, as well as fibrosis were found in HE staining section; The content of Tn-T and BNP in serum increased significantly(P<0.05).3. In the striatum of acute METH exposed rats, the content of NO and the activity of NOS are increased, and the content of protein with 3-nitrotyrosine was increased significantly (P<0.001). 4. There are 16 spots expressed differently in serum of rats, and ten of them are significant via tandem mass spectrum and data base search. There are three increased spots, and they are Glial fibrillary acidic protein, haptoglobin (Hp) and Carcinoembryonic antigen-related cell adhesion molecule 3 precursor. And there are six decreased spots, which are GTP-binding protein Rab-3D, Testis-specific serine kinase substrate, Ras-related protein Rab-3A, Sodium/hydrogen exchanger 3, Advanced glycosylation end product -specific receptor precursor and Alpha-1-inhibitor 3 precursor.CONCLUSION1. Both acute and chronic METH toxicity can cause obvious damage on heart, inducing the increase in heart rate and blood pressure, as well as the content of serum Tn-T. The long-time use of METH can increase the content of BNP, that means the heart function has been effected. While the short-time ones can not influence the heart function.2. METH can increase the content of NO and the activity of NOS in the striatum, then increase the content of protein with 3-nitrotyrosine, which may cause the damage or lose of protein functions. It may be one of the main mechanisms in METH-induced neurotoxicity.3. There are 16 spots expressed differently in serum of acute METH exposed rats, and ten of them are significant via tandem mass spectrum and data base search. These proteins mainly participate in cytoskeleton reconsitution, signal transduction pathway /transport/binding, and regulation of enzyme activity. The injury of these proteins may be the main mechanism of METH-induced toxicity. Additionally, the change of proteins in serum may be one of index to indicate METH-induced toxicity.更多还原