论文总字数：22363字

摘 要

本论文研究的目的是建立一种更加稳定、可靠的分析睾丸组织内源性代谢物的方法，通过分析雷藤甲素引起的雄性小鼠体内代谢变化，以研究其诱发的小鼠生殖毒性。本实验通过小鼠灌胃雷公藤甲素45 mg/kg/day诱发生殖毒性，检测其性腺指数、附睾精子数量和活动率、睾丸生化指标(ACP、LDH、SOD)，证明生殖毒性模型建立成功。本实验还建立了以GC-MS为基础的小鼠睾丸组织代谢组学的方法学研究和验证。用试剂盒、精子计数和GC-MS代谢组学标准方法分析血清和睾丸组织内的生物标志物。PCA、PLS-DA和GC-MS聚类分析方法分析血清和睾丸组织的多变量数据，从而区别正常组和模型组。将潜在代谢物的保留指数与内标物进行比较以确定其结构。雷公藤甲素诱发的体内变化包括糖酵解，三羧酸循环，脂肪酸代谢以及LDH和SDH能量代谢的改变。此外，进行血清分析和睾丸组织生物标志物的代谢反应对比可以更精确、更高效的确定模型组。我们的实验显示基于致病靶器官的代谢组学研究是分析传统中药鉴定潜在毒性的新兴技术。

关键词：雷公藤甲素 代谢组学 GC-MS 生殖毒性 精子

Abstrate

The purpose of this paper is to build a more stable and reliable method of analysis of endogenous metabolites in testicular tissue, and study the reproductive toxicity of triptolide-induced through the analysis the changes caused by the Triptolide in vivo metabolism of male mice. Model group mices were induced testicular injuries following oral administration 45 mg/kg/day Triptolide for 4 weeks. Subsequently, serum and testis tissue were analyzed for biomarkers using standard methods with commercial assay kits and sperm count as well as a metabolomic approach with GC-MS. PCA, PLS-DA and Hierarchical Clustering to the GC-MS based the serum and testis tissue metabolic profile revealed that model mice could be distinguished from normal animals. The structural identities of potential metabolites were confirmed using comparing retention index (RT)with mass spectral libraries or RT with the authentic standards. Metabolite changes associated with Triptolide induced testicular injuries progression are identified in the serum and testis tissue samples involved in several pathways, including glycolysis, the tricarboxylic acid cycle, fatty acid metabolism, cell membrane structure as well as LDH and SDH dependent energy metabolism pathway. In additional, compared with the serum profiling, metabonomic responses acquiring biomarkers on testicle tissues were higher accurate and specific in elaborating Triptolide induced-testicular toxicity. Our results indicated that metabonomic approach based on toxicity target organs would provide a useful tool for dissecting the potential toxicities and mechanisms of TCM.

Key words: metabolomics; triptolide; GC-MS ; testicular toxicity; spermatozoa

目录

摘 要 I

1.1中药雷公藤 1

1.2雷公藤甲素毒性研究 1

1.2.1消化系统毒性 2

1.2.2 循环系统毒性 2

1.2.3 泌尿系统毒性 2

1.2.4 免疫系统毒性 3

1.2.5 生殖系统毒性 3

1.3代谢组学 3

1.3.1谢组学的发展 4

1.3.2代谢组学的定义与分类 5

1.3.3 代谢组学的应用 5

1.4 药物代谢组学 6

1.5 代谢组学分析流程 7

第二章 实验部分 8

2.1前言 8

2.2 实验部分 9

2.2.1 动物实验 9

2.2.2 脏器指数检测 9

2.2.3 附睾精子数量及活动率检测 9

2.2.4 睾丸生化指标测定 9

2.2.5数据处理及统计分析 10

2.3 代谢组学研究 10

2.3.1 样品准备 10

2.3.2 GC-MS分析 10

2.3.3 数据采集处理及统计学分析 11

第三章 结果与讨论 12

3.1雷公藤甲素生殖毒性 12

3.1.1 体重和性腺指数 12

3.1.2精子数量测定 12

3.2代谢组学分析 13

3.2.1 GC-MS分析 13

3.2.2 数据分析 14

3.3讨论 21

第四章 小结 24

参考文献 25

致 谢 27

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