【作者】 王建华；

【导师】 李恩；

【作者基本信息】 河北医科大学 ， 中西医结合基础， 2003， 博士

【摘要】 目的：骨质疏松是一种全身性骨量减少，骨组织微细结构受损而导致骨脆性增加，骨折危险性升高的疾病。以单位的骨量降低，骨基质的有机成分生成不足，继发钙盐减少为特征。骨质疏松一般分为两大类：即原发性骨质疏松和继发性骨质疏松。前者又分为两型：绝经后骨质疏松(Ⅰ型)和老年性骨质疏松(Ⅱ)型。治疗骨质疏松的药物目前有三类：1、抑制骨吸收的药物：如雌激素及其类似物尼尔雌醇及新研制的利维爱，降钙素，二膦酸盐等；2、刺激骨形成的药物：如氟化物，同化类固醇激素等；3、作用于骨矿化的药物：如维生素D，钙剂等。 绝经后的妇女骨质疏松的发病率高达50％，因而是骨质疏松防治的重点。其发病与雌激素水平降低有关。因此雌激素是当前防治绝经后骨质疏松的首选药物，此疗法称为激素替代疗法(HRT)。雌激素在骨重建中起着非常重要的作用。骨重建过程包括旧骨的吸收和新骨的形成。成骨细胞和破骨细胞参与了这个过程的发生。成骨细胞和破骨细胞内都有雌激素受体。雌激素通过其受体发挥直接作用。一方面，促进成骨细胞增殖和胶原合成以及与骨矿化相关的蛋白的合成。另一方面，抑制破骨细胞的骨吸收，维持骨形成与骨吸收之间的平衡。此外，雌激素还通过钙代谢激素调节系统，影响三种钙代谢激素的分泌。雌激素还可通过下丘脑-垂体-性腺轴系统调节雌激素及受体的合成。通过上述三条途径雌激素防治绝经后骨质疏松。虽然雌激素替代疗法对身体有诸多好处，但最近的研究发现，15年以上的雌激素治疗患者，发生乳腺癌的危险性升高30％，并且雌激素的撤退可引起骨量丢失；另一个副作用是单独应用的雌激素有可能增加子宫内膜癌的发病危险。因此，开发研新的防治绝经后妇女骨质疏松的药物具有重要意义。 大豆异黄酮是从大豆中提取出来的异黄酮类化合物，具有弱的雌激素作用，因其来源于植物，而被称为植物雌激素。有学者认为，由于大豆异黄酮能选择性地与雌激素受体的β亚型(ERβ)结合，所以无应用中文摘要雌激素所带来的副作用。其作用与体内的雌激素水平有关，对低雌激素水平者，可与雌激素受体结合，而发挥雌激素样作用;对高雌激素，可与雌激素竞争雌激素受体，而表现抗雌激素作用。因而可用于治疗与雌激素相关的一些疾病，如骨质疏松及高血脂症等。 目前对大豆异黄酮防治骨质疏松的研究，多用含大豆异黄酮的蛋白或粗提取物进行，而用纯的大豆异黄酮的很少，且实验结果有争议，有的研究结果认为，大剂量大豆异黄酮有效，有的认为大剂量无效而小剂量有效。 植物雌激素除异黄酮外，尚有木脂素和香豆素两类，有趣的是，某些补肾中药含有这两类物质。这两类物质是否是某些补肾中药补肾作用的物质基础呢?中医认为“肾主骨生髓”，所以本研究也同时探讨某些补肾中药如五味子及蛇床子中所含有的木脂素和香豆素对骨代谢的影响。 方法:基于上述理论，采用整体动物实验与离体细胞培养实验相结合的方法，探讨大豆异黄酮防治去卵巢大鼠的骨质疏松的作用及其作用机制。 动物模型，选用6月龄SD大鼠进行卵巢切除造成骨质疏松模型。 选用含量达90%的大豆异黄酮给予动物，设大小两个剂量，同时设尼尔雌醇组作为阳性对照。 动物实验部分，选择血清总碱性磷酸酶(ALP)骨碱性磷酸酶仍ALP)、骨钙素(BGP)等骨形成指标，同时选择抗酒石酸酸性磷酸酶(T RACP)、尿钙与尿肌配的比值(C盯Cr)，尿经脯氨酸与尿肌配的比值(HOP/Cr)等骨吸收指标。观察大豆异黄酮对骨吸收和骨形成的影响，探讨其作用机制。 测定大鼠股骨和胫骨的骨密度(B MD)，以观察其疗效。为了考察大豆异黄一酮对雌激素的影响，同时测定了血清雌二醇的水平。 I型胶原是成骨细胞增殖分化时合成的胶原蛋白，占骨有机质的80一90%，为了从分子水平探讨大豆异黄酮的作用机制，采用RT一PCR及免疫组化的方法，测定股骨I型胶原表达的变化。 细胞实验部分，用大豆异黄酮中的两个单体成分一染料木素和大豆贰元进行离体实验，观察其对大鼠成骨细胞增殖分化的作用，同时采用RT一PCR及免疫组化的方法，观察其对I型胶原表达的影响。同时观察................ 2 中文摘要补肾中药五味子中的五味子甲素及五味子乙素等木脂素类植物雌激素，及蛇床子中的蛇床子素(属香豆素类植物雌激素)对成骨细胞的增殖分化的作用，成骨细胞分化的指标选用碱性磷酸酶(ALP)或骨钙素(BGP)。 将不同的中药单体成分加入培养的大鼠成骨细胞体系，测定其对成骨细胞增殖分化的影响，从细胞水平探讨其作用机制。 预期结果:比较大豆异黄酮与雌激素类药物的疗效，确定有效剂量，并探讨其可能的作用机制，找出某些补肾中药补肾作用的物质基础，实验分为五个部分。 结果:1大豆异黄酮对去卵巢大鼠骨质疏松的防治作用 采用6月龄SD大鼠，按体重随机分为5组，即假手术组(S FIAM)，去卵巢组(OVX)，阳性对照组(EZ)，更多还原

【Abstract】 Objective: Osteoporosis is a disease of bone metabolism of the whole human body. The bone mineral density is lower and the structure of bone is changed .In osteoporosis patients, in addition, the risk of fracture is higher for the bone toughness of the patients is decreased. At present, osteoporosis is common in old people and menopause woman. Its incidence increases with aging, 20% in man, 50% in menopause women.There are two kinds of osteoporosis in general: original contracted osteoporosis and contracted osteoporosis due to other diseases. There are two types of the first kind: post-menopausal osteoporosis ( I) and aging osteoporosis (II). There are three kinds of medines used for the treatment of osteoporosis: first group, those which can inhibit the bone absorbing process, such as E2, nilestriol, CT, etc. The second group are those which can promote bone forming , such as Tridin, PTH etc. The last group consist of the medicines which can benefit the mineral deposit process, such as alfacalcidol, caltrate D600 etc.Much attention is paid to post-menopausal osteoporosis because its cause is clear, that is, due to lower estrogen level in the body. Estrogen replacement therapy (ERT) is widely used in preventing post-menopausal osteoporosis. But, this method may increase the risk of developing breast cancer . Many women do not like it. So, to explore a new medicine is of importance.Phytoestrogens are some compounds obtained from plants, Structurally, they share many similarities with endogeneous estrogens, they can bind to estrogen receptors, and they may exert both estrogenic and antiestrogenic effects depending on circulating levels of endogenous sex hormones. Soybean isoflavones are compounds obtained from soybean.They exhibit weak estrogenic activity on the order of 10~2-10~3 that of 17 beta -estradil ; but may be present in the body in concentrations 100-fold higher than endogenous estrogens. But they have little side effects . They have been shown to be effective in promoting bone mass and preventing bone loss.Ligant and coumarins are also phytoestrogens. Some traditional Chinese medicines contain those compounds. According to Chinese medicine, " kidney controls the bone". And those medicines are tonifying kidney recipe. Maybe those compounds have some effects on bone.Based on the above theories, the effects of phytoestrogen, particularly isoflaveones, on osteoporosis in ovariectomized rats and their mechanisms were studied.Method: Six-month SD femate rats were divided randomly into 5 groups: (1) SHAM group (control group); (2) OVX (ovarectomy); (3) H-ISO (ovx+soy isoflavones 30 mg ·kg-1) ; (4) L-ISO (ovx+soy isoflavones 15 mg · kg-1); (5) E2 (ovx+nilestriol).Tibia and femur bone mineral density (BMD) of rats were measured by using dual-energy X-ray absorptiometry.ALP, BALP were measured to observe the effects of soy isoflavones on the bone builting process. To observe the effects on the bone resorping process, Ca/Cr, HOP/Cr and TRACP were measured.The level of serum E2 and BGP were measured by RIA. RT-PCR and immunohistochemistry were used separately to semi-quantify the amount of collagen type I of rat femur and osteoblasts to observe the changes caused by soy isoflavones.MTT and PNPP were used separately to observe the proliferationand the activity of ALP of osteoblasts cultured in vitro with soy isoflavones( such as Genistein, Daidzein), or lignans ( such as deoxyschizandrin andgamma-schizandrin ) or coumarins (such as osthol) of different concentrationin it for different incubation periods .The whole experiments consist of 5 parts:1 The preventive effects of soybean isoflavones on ovariectomized rat osteoporosisSix-month SD femate rats were divided randomly into 5 groups: (1) SHAM group (control group); (2) OVX (ovarectomy); (3) H-ISO (ovx+soy isoflavones 30 mg·kg-1); (4) L-iso (ovx+soy isoflavones 15 mg·kg-1); (5) E2 (ovx+nilestriol).Animals in the SHAM group were subject to sham operation .Ovariectomies were performed in other gr更多还原