【作者】 张国华；

【导师】 王保捷；

【作者基本信息】 中国医科大学 ， 法医学， 2003， 博士

【摘要】 研究目的 药物滥用是国内外突出的社会和医学问题。海洛因、吗啡、度冷丁是目前我国滥用的主要毒品种类。长期药物滥用引起吸毒者药物依赖和成瘾及神经系统损害。 阿片类毒品长期滥用可引起神经细胞变性、坏死，有的形成白质脑病，病变分布于丘脑、纹状体、中脑导水管周围灰质、大脑皮层及苍白球，与阿片受体脑中枢密集分布的部位基本吻合。阿片类毒品引起的神经细胞的死亡是否伴有凋亡的发生，以及神经细胞的凋亡与药物依赖和成瘾机制的关系有待于进一步研究。 一氧化氮(NO)是一种自由基气体，具有广泛的生物学效应，对神经元有毒性作用。海洛因滥用者血清中NO含量明显增高。神经性结构型一氧化氮合成酶(ncNOS)是脑神经细胞NO生成的关键酶。ncNOS、caspase-3、Fas、Bax蛋白因子与细胞凋亡有关，其表达增加可引起该细胞的凋亡。 虽然国内外学者对相关内容进行了一些研究，但有些内容的研究工作刚刚起步，尚存在一些重要问题需进一步研究和探讨。 1．海洛因、吗啡、度冷丁慢性依赖和自然戒断对脑组织的损害，尤其与阿片类药物滥用关系密切的中枢神经系统重要功能部位大脑皮质、中脑导水管周围灰质和中脑腹侧被盖区神经细胞的损害； 2．海洛因、吗啡、度冷丁慢性依赖和自然戒断是否引起脑神经细胞的凋亡； 3．海洛因、吗啡、度冷丁慢性依赖和自然戒断脑神经细胞ncNOS、caspase-3、Fas、Bax蛋白免疫组织化学变化和ncNOS mRNA、caspase-3 mRNA原位分子杂交基因的表达； 4．海洛因、吗啡、度冷丁三种滥用药物对中枢神经系统损害程度的比较； 5．探讨海洛因、吗啡、度冷丁药物依赖和神经生物学损害的机制； 6．探讨阿片类药物滥用脑神经细胞ncNOS上aspase－3、Fas和Bax免疫组织化学变化特点及法医学鉴定的诊断价值。 实验材料与方法 1．动物实验过程：将35只SD大鼠（体重200－2509入随机分为7组，每组5只。分组如下（实验过程中，9只动物死亡未计算组内卜 C组：等容积生理盐水皮下注射对照组 HI组：海洛因慢性依赖组 HZ组：海洛因慢性依赖后自然戒断组 MI组：盐酸吗啡慢性依赖组 MZ组：盐酸吗啡慢性依赖后自然戒断组 DI组：度冷丁慢性依赖组 DZ组：度冷丁慢性依赖后自然戒断组 大鼠在自然光线充足在5℃左右、单笼适应性饲养Zd后开始实验。每日称重。以剂量递增法经皮下注射给药 60d，海洛因起始剂量 Zing·kg－‘·d－‘，每 sd递增 ling·kg－‘，递增至 13mg·kg－‘·d－’；盐酸吗啡起始剂量为20mg·kg’‘·d’‘，每sd 递增 10mg·kg－‘，递增至140mg·kg－’·d’‘；度冷丁起始剂量为 20mg·kg－‘·d＊，每 sd递增 10mg·kg－’，递增至 140mg·kg－’·d－‘。每日剂量分 3次给药，给药间隔超过 6h （：30，13：30，19：30入建立药物慢性依赖动物模型。慢性依赖组和对照组至规定时间处死动物、连续灌流固定、切取大脑、中脑组织块。自然戒断组停药，连续观察4d，称体重，记录戒断症状，处死动物、灌流固定、取材。 2．检测方法 O）常规石蜡切片刀．E．染色、观察； p）超微结构观察：透射电镜标本常规制备，超薄切片，铅铀双染色，H600－TEM日立透射电子显微镜下观察和拍照实验结果； 橱）免疫组织化学染色：采用SP法对ncNOS／aspase－3＊as和Bax进 ·2·行染色； （4）原位杂交染色：ncNOS m血A和 caspase－3 mRNA； K）细胞凋亡染色：TUNEL标记法； 忙）图像分析：应用 Motto IInages Advanced 3．口图像分析系统对免疫组织化学染色及原位杂交的阳性反应产物进行灰度检测和阳性细胞数计算。 3．数据的统计学处理 采用SAS统计软件对实验数据进行统计学处理，数据应用均数土标准差（XSSD）表示，多组间比较应用ANOVA法进行方差分析，各实验组与对照组比较应用Dunnett t检验，两两组间比较应用Student－Neman－Keuls＜检验进行统计。P＜0．05表示检验分析有显著性差异。 主要实验结果 1．海洛因、吗啡、度冷丁慢性依赖和自然戒断后大鼠的表现 慢性依赖组开始表现为呼吸不平稳、促快或浅慢、竖尾，以后逐渐表现为给药后出现明显兴奋症状，跳跃、竖尾等。自然戒断组停药10－12h逐渐出现明显的戒断症状和体征：烦躁不安、高度激惹、蹦跳、尖叫、异常姿势J齿、咬牙、湿狗样颤抖、甩头、上睑下垂、腹泻、清理皮毛、站立等，体重也明显下降。戒断24—36h最为明显。以后逐渐减弱，戒断96h症状基本消失。海洛因实验组表现最为明显，依次为吗啡和度冷丁实验组。 2．海洛因、吗啡、度冷丁慢性依赖和自然戒断大鼠脑组织病理形态学改变 门）脑宏观检查未见明显异常。 p）H．E．染色：脑水肿，有的神经细胞嗜伊红染色增强、均质变、萎缩；有的神经细胞淡染，尼氏体消失或膜下积聚。 瞩）神经细胞?更多还原

【Abstract】 ObjectiveDrug abuse is obvious social and medical problem at home and abroad. Heroin, morphine and pethidine are main poisons in our country at present. Long - term drug abuse will result in drug dependence, addiction and damage in nervous system.Long - term drug abuse with opiums poisons may cause degeneration and necrosis in nerve cells. There are encephalopathy in white matter in some cases. These pathological changes are in thalamencephalon, striatum, periaqueductal gray matter, cerebral cortex and pallidum, distribution of which are coincided with location of opiate receptors in central nervous system. It need further researching that whether neurocytes death in drug abuse is accompanied with apop-tosis and what s relation between neurons apoptosis and mechanism of drug dependence and addiction.NO is a free radical gas, which posses wide biological effects and has toxic effect to neurons. NO in blood serum of heroin drug abuser is high clearly. Neu-ronal constructive nitric oxide synthase(ncNOS) is a key enzyme in NO production, caspase -3, Fas and Bax protein are related to apoptosis. Increases of nc-NOS, caspase - 3, Fas and Bax content in neurons will cause apoptosis.There are many important matters which need more investigating and discussing.1. Damages of brain tissue by heroin, morphine and pethidine chronic dependence and spontaneous withdrawal, especially in neurons of cerebral cortex, periaqueductal gray matter and the ventral tegmental area which are closely related to opiums drug abuse;2. Whether do heroin, morphine and pethidine chronic dependence and spontaneous withdrawal cause neurons apoptosis in brain?3. Changes of ncNOS, caspase - 3 , Fas and Bax by immunohistochemistry and expresses of ncNOSmRNA and caspase -3mRNA by in situ hybridization in brain neurons by heroin, morphine and pethidine chronic dependence and spontaneous withdrawal;4. Impaired degrees in central nervous system by heroin, morphine and pethidine;5. Mechanisms of lesions in neurobiology and drug dependence by heroin, morphine and pethidine;6. Distinguishing feature and diagnostic value of immunohistochemical changes of ncNOS, caspase - 3, Fas and Bax in brain neurons by opiums drug abuse.Materials and MethodsBased on these bases and questions described above, in this study, the model of heroin, morphine and pethdine chronic dependence and spontaneous withdrawal in rats was built. 35 male Sprague - Dawley rats (200 - 250g at the start of experiments) were caged individually and allowed to habituate to the environment 2 days. The animals were randomly divided into control ( C) , heroin chronic dependence ( H1 ) , heroin spontaneous withdrawal ( H2 ) , morphine chronic dependence ( Ml ) , morphine spontaneous withdrawal ( M2 ) , pethdine chronic dependence(D1) , pethidine spontaneous withdrawal (D2) groups, each consisting of 5 animals. Seven groups were treated with saline, heroin, mor-phine or pethidine by subcutaneous injection (s. c. ) for 60 days. Dose of heroin was from 2mg kg-1 d-1 to 13mg kg-1 d-1. Morphine, from 20mg kg-1 d-1to 140mg kg-1 d-1 Pethdine, from 20mg kg-1 d-1to 140mg kg-1 d-1. Treatment interval was 6 h (7:30, 13:30, 19:30). Following 60 days of administration, when rats were completely chronic dependence to the opiums drugs, rats of C, H1, Ml and D1 groups were fixed by instillation with formaldehyde fixation solution and whole brains were removed. Natural opiate withdrawal was induced by ceasing opiums drugs administration in H2, M2 and D2 groups. Rats were observed for 4days. Then, the animals were killed according to method described above.The chosen other experimental methods were as follows:1. Conventional H. E. staining.2. Ultrastructural observation by transmission electron microscope.3. ncNOS, caspase-3, Fas and Bax immunohistochemistry by SP method.4. In situ hybridization of ncNOSmRNA and caspase - 3mRNA.5. Terminal deoxynucleotidyl transferase - mediated dUTP nick end labeling (TUNEL).6. Imag更多还原