论文总字数：22997字

摘 要

血管新生(angiogenesis)，是指从已存在的血管中新生出微血管的过程。血管新生对于组织再生和修复起着重要作用，对缺血性心脑疾病、肢体缺血、创伤修复等缺血性疾病治疗极为重要。寻找和发现具有促进血管新生作用的单体化合物和复方制剂，并阐明其作用机制，已成为研究人员关注的热点。但是由于传统的血管新生研究模型存在无法实时观察血管新生这一复杂、动态的体内生理过程，因此，近年来斑马鱼作为新型的血管新生研究模型已成为促血管新生活性物质研究的有力工具。

甘草作为常用中药材，传统中医理论认为甘草具有补脾益气，清热解毒，调和诸药的功能，现代药理研究表明甘草具有多种药理活性，其中的黄酮类成分是其重要的药效物质基础，但甘草的各类成分在血管新生方面的研究报道较少。因此，本论文选择了甘草中的两个黄酮类单体甘草苷、异甘草苷作为研究对象，在应用斑马鱼体内血管新生促进模型进行活性筛选后发现，异甘草苷具有较为显著的促进血管新生活性，而甘草苷促血管新生活性较弱。进一步在人脐静脉内皮细胞(HUVECs)模型上验证，实验结果显示异甘草苷对细胞增殖无明显抑制作用，并能在一定浓度内剂量依赖性地促进细胞增殖，划痕实验结果显示异甘草苷能促进HUVEC细胞划痕损伤后的迁移。

关键词：血管新生 甘草苷 异甘草苷 斑马鱼

Research of liquiritin and isoliquiritin on promoting angiogenesis

Abstract

Neovascularization(angiogenesis) is the process of capillaries newborning from pre-existing the blood vessels,which is very important for tissue regenerationand repairation.And it plays great role in cerebralis chemic heart disease,limb ischemia,ischemic wound healing,and other disease treatment.Seeking the monomer compounds and compound preparation which can promote angiogenesis and clarifying its mechanism of action has become the focus of attention of researchers.However,the traditional model of angiogenesis research can not observe complex,dynamic physiological processes in vivo in real time.Therefore,in recent years,the zebrafish as a new model of angiogenesis research has become a poweful tool for pro-angiogenic activity of research.

Licorice as common herbs,traditional Chinese medicine theory holds that licorice has the function of detoxification, spleen Qi and reconciling the various drugs.Modern pharmacological studies have shown that licorice has a variety of pharmacological activities,and flavonoids is an important material basis of efficacy.But the reports about many kinds of licorice ingredient in angiogenesis research are fewer.Therefore, this paper chose two flavonoid monomers in licorice liquiritin and ,isoliquiritin as the research object.After the application of the zebrafish model to promote angiogenesis in vivo activity screening,it is found that isoliquiritin has a more significant role in promoting angiogenic activity, while liquiritin is weak.Further on human umbilical vein endothelial cells (HUVECs) model validation, experimental results show that isoliquiritin has no significant inhibitory effect on cell proliferation and promote cell proliferation can be dependent on the dose of a certain concentration, scratches experimental results show that isoliquiritin obviously promote migration of HUVEC scratch injury.

Keywords: angiogenesis; liquiritin; isoliquiritin; zebrafish

目 录

摘要.................................................................................................................................................I

Abstract…........................................................................................................................II

第一章 综 述……………………………………………………………..………………….1

1.1 药物简介 2

1.1.1 国内外研究情况 2

1.1.2 甘草中黄酮的药理活性研究进展........................................................3

1.1.3 临床研究 4

1.2 血管新生简介 4

1.2.1 血管新生相关机制 4

1.2.2 临床意义 5

1.3 促血管新生的研究模型 6

1.3.1 鸡胚尿囊膜研究模型............................................................................6

1.3.2 斑马鱼的研究模型 6

1.3.3 HUVEC人脐静脉内皮细胞的研究模型 7

第二章 异甘草苷与甘草苷对斑马鱼血管新生活性的研究 8

2.1 实验仪器与材料 8

2.1.1 实验材料 8

2.1.2 主要实验溶液和药物的配制.................................................................8

2.1.3 斑马鱼胚胎的培养 9

2.2 实验方法 9

2.2.1 血管受损模型实验方法 9

2.3 实验结果与讨论 10

2.3.1 异甘草苷和甘草苷恢复VRI诱导转基因斑马鱼受损血管生成 10

第三章 异甘草苷与甘草苷在HUVEC细胞模型活性评价…………............13

3.1 实验仪器与材料 13

3.1.1 实验材料 13

3.1.2 主要试剂与耗材..................................................................................13

3.1.3 主要仪器 14

3.1.3 体外血管新生抑制活性的检测及机制研究方法 15

3.2 实验结果与讨论 17

3.2.1 CCK-8细胞增殖活力检测实验结果....................................................17

3.2.1 HUVEC细胞迁移实验结果 ..18

参考文献…....................................................................................................20

致谢…............................................................................................................22

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