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1、摘 要目的：藜芦为百合科藜芦属植物。南方主要有毒植物记载，藜芦根茎叶花均可以引起中毒，地下部分的根茎部毒性为最高。导致中毒的症状为：腹部热痛，流涎，呕吐，恶心，腹泻，出汗，虚弱，丧失意识；严重中毒出现脉率异常，大便出血，浑身抽搐，昏迷，最后因停止呼吸而致死。藜芦生物碱是藜芦的主要活性成分，同时又是毒性成分。藜芦生物碱具有显著降压和抗肿瘤等活性，其毒性主要是对神经系统和胚胎有影响。表现为引起神经系统的钠离子通道和钙离子通道持续放电，以及导致多种实验动物致畸现象。由于前期发现藜芦生物碱可引起小鼠脑神经DNA损伤，结构特征不同，损伤程度也有差异。所以，本课题以藜芦生物碱为研究对象，探讨藜芦生物碱不同
2、结构特征的单体成分对小鼠脑细胞DNA损伤的影响，建立构毒关系，以期为藜芦进一步的毒理研究提供理论依据。方法：细胞DNA损伤与修复、DNA交联、放射生物学及遗传毒理等领域的研究，广泛用彗星实验（单细胞凝胶电泳实验）在单细胞水平上检测DNA损伤。本实验采用彗星实验的方法检测19种不同结构特征的藜芦生物碱对小鼠小脑和大脑皮层脑细胞DNA损伤作用，并且分为不同剂量组给药，通过实验数据来比较其损伤程度，探讨藜芦生物碱引起小鼠脑细胞DNA损伤的结构与毒性的关系。结果：用CASP软件对彗星实验所得的细胞DNA损伤图分析，对数值进行汇总得出：母核结构为西藜芦碱类和介藜芦胺类，对DNA损伤基本强于母核结构为维藜
3、芦碱类、番茄立定碱类；介藜芦胺类结构中D环为苯环结构的藜芦胺（Veratramine）引起DNA损伤显著，藜芦胺结构3位葡萄糖苷化的藜芦新碱（Veratrosine）引起DNA损伤较显著；西藜芦碱类结构中有12位羟基取代的12-羟基藜芦酰棋盘花碱（12-hydroxylveratroylzygadenine）引起DNA损伤显著。结论：藜芦生物碱对小鼠小脑和大脑皮层细胞DNA均有不同程度的损伤作用；大部分呈剂量依赖性。不同母核特征的藜芦生物碱对小鼠小脑和大脑皮层细胞DNA损伤程度有差异，并且同一母核特征的生物碱所连的取代基团结构对损伤程度也有直接的关联性，为后期深入探讨其致毒机理提供理论依据。关
4、键词：藜芦生物碱，彗星实验，DNA损伤，构毒关系39ABSTRACTObjective: Liliaceae Veratrum plant.South“Poisonous Plants”records,quinoa phragmites flower stems can be caused by poisoning,toxic roots underground part of the Ministry of the highest.Lead poisoning symptoms are:abdominal pain,fever,salivation,vomiting,nausea,diarr
5、hea,sweating,weakness,loss of consciousness;severe poisoning appear abnormal pulse rate,bowel bleeding,covered with convulsions,coma,and finally stopped breathing due to death.Alkaloids is the main active ingredient hellebore,but it is also toxic ingredients.Alkaloids have significant antihypertensi
6、ve and anti-tumor activity,its toxicity primarily affects the nervous system and the embryo.Performance of the nervous system caused by sodium ion channels and calcium channels continuous discharge,and causes avariety of experimental animals Teratogenicity phenomena.Since the early discovery,alkaloi
7、ds can cause DNA damage in mouse brain,different structural characteristics,there are differences in the degree of injury.Therefore,subject to the alkaloids for the study,investigate the effect of alkaloids different structural characteristics of monomer components of DNA damage in mouse brain cells
8、 to build structures poison relations,with a view to further toxicological studies provided hellebore theory.Methods: The study of DNA damage and repair,DNA crosslinking, radiation biology and genetic toxicology and other areas,widely used comet assay (single cell gel electrophoresis) to detect DNA
9、damage at the single cell level.The experiment using comet assay was used to detect 19 different structural characteristics of alkaloids mouse cerebellum and cerebral cortex of DNA damage in brain cells,and is divided into different dose group,to compare the extent of damage to the experimental data
10、,explore alkaloids cause DNA damage in mouse brain cells relationship between structure and toxicity.Results: CASP software for cells DNA damage resulting figure comet assay analysis,summarize numerical results:core structure for the West hellebore hellebore referral bases and amines,DNA damage in t
11、he nucleus of a strong basic structure for peacekeeping hellebore bases,tomato standing bases;referral hellebore D amine structure veratramine benzene ring structure caused significant DNA damage,veratramine 3 glucoside structure of veratrosine induced DNA damage more significant;West Veratrum alkal
12、oids structure has 12 hydroxyl-substituted 12-hydroxy acid checkerboard hellebore flower base (12-hydroxylveratroylzygadenine) induced DNA damage is significant.Conclusion: alkaloids on mouse cerebellum and cerebral cortex cells have DNA damage of varying degrees;most dose-dependent manner.Different
13、 characteristics of the nucleus are different alkaloids and extent of the cerebellar cortex cell DNA damage in mice,and features the same nucleus of alkaloids substituents attached structure on the extent of damage also has direct relevance for post depth To investigate the mechanism of its toxicity
14、 to provide a theoretical basis.Keywords: veratridine,comet assay,DNA damage,Structure poison relationship目 录摘 要IABSTRACTIII第一章 前言1一 藜芦介绍111藜芦化学成分研究进展112 藜芦药理研究进展313 藜芦毒性和毒理研究614 藜芦生物碱的代谢研究815 藜芦的中药配伍研究进展916 藜芦的构毒关系研究1017 本节小结11二 单细胞凝胶电泳实验介绍1121 DNA损伤、检测方法1122 SCGE 实验发展简介1223 SCGE实验方法1324 SCGE实验的应用1
15、7三 选题依据和研究意义18第二章 藜芦生物碱引起小鼠脑细胞DNA损伤的构毒关系研究21一实验材料2111 实验动物2112 试剂与药品2113 实验仪器2314 分析软件24二 实验方法2421 试剂的配制2422 样品给药2423 细胞混悬液的制备2424 玻片的制备2425 细胞的裂解2526 DNA的碱解旋2527 电泳2528 中和、固定2529 染色、阅片26210 统计分析26三 实验结果2631 实验方法的建立2632 实验结果数据及作图27四 结论和讨论3541 不同藜芦碱引起小鼠脑部DNA损伤3542 SCGE实验方法简议36第三章 全文总结39参考文献41攻读学位期间发表的学术论文目录51致 谢53第一章 前言一 藜芦介绍百合科植物藜芦，全世界大概有40种，分布在欧洲、北美和亚洲，中国植物志整理记载，在我国藜芦属植物大约24种，其中发现有药用植物的有13个种和1个变种。在上世纪末期，四川、云南地区出现2个新品种有： 贡山藜芦和南川藜芦1；发现于秦岭的有一个变种：总状藜芦2。藜芦属植物在我国境内分布以云、川地区分布最多，东北、东南、中原地区也有分布。常用的药用植物有：藜芦、蒙自藜芦、兴安藜芦、狭叶藜芦、尖被藜芦等3。藜芦味辛，苦，性寒，有大毒，归胃，肝，肺经。中国传统医学里藜芦用药早在神农本草经就有记载，用药部位在根
