卷柏化学成分及雌激素样作用机理研究

【作者】 郑晓珂；

【导师】 王继峰；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2006， 博士

【摘要】 雌激素(estrogen)是女性关键性的调节激素,在体内具有广泛的生物学活性,不仅对生殖系统具有重要的调控作用,而且对骨质代谢和脂类等多种代谢也有着重要的调节作用。女性进入绝经期后,随着卵巢功能衰退,体内雌激素水平下降,在出现更年期综合症的同时,心血管疾病、动脉粥样硬化和骨质疏松性骨折等疾病的发病率也随之增高。长期以来一直认为雌激素替代治疗可缓解妇女绝经期综合征症状,减慢动脉粥样硬化进展、减少骨质丢失从而降低骨质疏松性骨折的发生率。然而,随着近年来的研究发现,长期使用雌激素具有一系列严重的副作用:长期服用会增加女性中风、血管栓塞、心脏病发作、痴呆的发病率,特别是有诱发子宫内膜癌、乳腺癌的危险。植物雌激素是一类存在于植物中,与雌激素结构相似的一类生物活性物质。在体内与雌激素受体(estrogen receptor、ER,ERα和ERβ)结合,具有双向调节作用:即当体内雌激素水平较高时植物雌激素发挥抗雌激素活性,当体内雌激素水平较低时植物雌激素具有拟雌激素作用,且没有明显的人工合成雌激素的副作用。我国地域辽阔,中药资源极其丰富,许多特有的药用植物主要分布在我国境内,祖国医学经历长期临床实践,积累了应用植物药防治疾病的丰富经验,但对中药雌激素方面的实验研究相对还较薄弱。从中药中寻找天然、副作用小的植物雌激素,进一步阐明中药的作用机理,将会指导临床医生在处方配伍时对疗效及安全性进行综合考虑。根据体内的雌激素信号转导途径,我们选用了以luc为报告基因、ERE为调控序列的重组报告基因载体pERE-TAL-luc,并将其与重组人ERα(humanERα,hERα)表达载体pCXN2-hERα和重组人ERβ(humanERβ,hERβ)表达载体pCXN2-hERβ共转染ER阴性细胞系,建立了靶向ERα或ERβ的细胞水平药物筛选模型,结合小鼠子宫增重实验,对卷柏、松针、益母草、石胆草及贯众等5种中药的植物雌激素作用进行了研究,以期为临床防治更年期综合症等提供准确、科学的实验基础。论文主要研究内容和结果如下:1.卷柏等5种中药植物雌激素活性筛选的实验研究采用小鼠子宫增重实验结合荧光素酶报告基因技术,对卷柏、松针、益母草、石胆草及贯众等五种中药进行了植物雌激素活性的研究。结果:卷柏总提取物能增加性未成熟小鼠子宫系数,可使小鼠子宫重量增加(P<0.01);细胞实验证明卷柏可通过hERα或hERβ诱导受ERE调控的报告基因luc的表达(P<0.01),表明卷柏通过ERα或ERβ发?挥雌激素样作用。卷柏与ERα的亲和力较ERβ的亲和力大,说明卷柏激活基因转录主要是通过ERα介导的,与文献报道植物雌激素主要作用于ERβ的普遍倾向有所不同。而松针、石胆草、益母草、贯众不能增加性未成熟小鼠子宫系数,故未进行进一步实验。更多还原

【Abstract】 Estrogens are vital regulation hormone for women and have broad range of biological activities. Estrogens play important roles in both the function of the reproductive system and the female mammary gland and so-classical target tissues, such as the brain, bone, liver, kidney, and cardiovascular system. As the level of estrogen decreases in postmenopausal women incidences of fracture in osteoporosis, cardiovascular and neurodegenerative diseases increase.Estrogens replacement therapy is able to attenuate symptoms in menopause syndrome, reduce incidence of above-mentioned diseases. However, studies find that long term use of estrogens also increase the risk of stroke, blood clots, heart attacks and dementia and particularly breast and uterine tumor in postmenopausal women.Phytoestrogens are the compounds capable of binding estrogen receptors which have dual actions. They increased the levels of estrogens when in low estrogens levels, in the other hands; they reduced the levels of estrogens when in high estrogens levels.China is vast in territory, with the extremely rich resources of Chinese herbal medicine, and many unique medicinal plants only distribution in China, the country’s long-term clinical medical practice and accumulated rich experience in the application of plant disease control, but study on phytoestrogens is rare. Search for natural, no side effect plant estrogenic from Chinese herbal medicine and further clarifying the role of Chinese herbal medicine mechanisms will guide clinicians to consider the efficiency and safety when prescribing.According to the classical signal transduction pathway of estrogen, we use Luciferase reporter gene to screen phytoestrogen form Selaginella tamariscina, Pinus massoniana Lamb, Corallodiscus flabellate, Dryopteris sublaeta Ching et Hsu and Leonurus heterophyllus. In this study,the pβgal-Control vector, the pERE-TAL-luc vector, the pCNX2-hERα/pCNX2-hERβvector were transfected into HEK293 cell line. If the components in the herbs combine with the ER, then it could induce the expression of reporter gene controlled by ERE.It is found that the water extract part and n-butanol extract part of Selaginella tamariscina are the active parts.1. The experimental study of estrogenic activities of five kinds of Chinese herbal medicinesUse uterus growth test and reporter gene technique to study the estrogenic activity of Selaginella tamariscina, Pinus massoniana Lamb, Corallodiscus flabellate, Dryopteris sublaeta Ching et Hsu and Leonurus heterophyllus Sweet.Result: The botanical extracts of Selaginella tamariscina can increase the uterine weights of mice (P<0.01); In the expression of reporter gene controlled by ERE, Either ERαor ERβ’s更多还原