【作者】 付雨；

【导师】 吴克明；

【作者基本信息】 成都中医药大学 ， 中医妇科学， 2010， 博士

【摘要】 目的：在中医理论指导下,参照前期自主研究的小鼠模型,建造稳定可靠的雷公藤多苷致大鼠卵巢功能低下模型；运用“以药测证”法探讨该模型的中医证候属性及补肾中药复方干预性治疗的作用机理,为中医药调节女性生殖轴的药效学机理研究提供一个新的符合中医证候特点的病证结合动物模型。方法：将雌性育龄期SD大鼠行阴道脱落细胞学涂片,筛选出动情周期正常者为受试动物。随机选取6只为正常对照组,其余大鼠参照前期方法予雷公藤多苷40mg/kg/d灌胃造模,同时监测动情周期的变化,当全部动物均出现动情周期紊乱时,确定造模成功。将已造模的30只大鼠随机分为5组,每组6只,分别为模型组、倍美力组、中药高剂量组、中药中剂量组、中药低剂量组,分别予倍美力和通脉大生片不同剂量治疗7天后(正常对照组同期给予等量生理盐水),处死动物。观察大鼠一般情况,称重法测算卵巢指数；放射免疫法检测血清雌二醇、孕酮；酶联免疫吸附法检测抑制素、B水平；免疫组化法检测卵巢雌激素受体(ER)、孕酮受体(PR)、肿瘤坏死因子及其受体(TNF-a/TNFR-1)、Caspase-3、血管内皮生长因子及其受体(VEGF/Flk-1)、内皮抑制素(Endostatin)等；将卵巢组织病理切片HE染色后在光学显微镜下观察分析卵巢组织细胞学形态改变并计数各期生长卵泡数与黄体数。结果：(1)予雷公藤多苷40mg/kg/d灌胃,连续10周,可造成雌性大鼠卵巢功能低下状态,表现为动物一般情况差,动情周期紊乱,卵巢指数下降；血清E2、INHB水平明显降低；卵巢组织中ER表达明显降低,细胞凋亡因子TNF-a、Caspase-3表达明显升高；卵巢血管中VEGF表达显著降低,血管生成抑制因子Endostatin表达显著升高。卵巢中生长卵泡数量明显减少,且卵巢血管壁变厚,管腔变窄。(2)运用通脉大生片干预性治疗后,大鼠一般情况得到明显改善,动情周期恢复至正常范围,卵巢指数升高；血清E2、INHB水平明显升高；卵巢血管中VEGF及其受体的表达明显升高,而Endostatin表达明显降低；卵巢中细胞凋亡因子TNF-a、Caspase-3表达明显降低；卵巢中生长卵泡数量明显增多,且卵巢血管壁变薄,管腔增宽。结论：①雷公藤多苷可造成雌性大鼠卵巢功能低下,补肾气益精血的中药复方“通脉大生片”可通过提高激素水平,增强卵巢局部血供,抑制卵巢细胞凋亡等方面,有效地改善和提高卵巢功能。②补肾中药复方治疗的有效性在一定程度上证实了该大鼠模型符合中医肾虚证候属性。更多还原

【Abstract】 OBJECTIVE:In order to have better animal models with required TCM syndrome characters for pharmacodynamic research in the effects of TCM treatment on female reproductive system, based on TCM theories and our previous research in making mouse models, Tripterygium Glycosides is given to rats to make a repeatable and reliable model of ovarian hypofunction. Method of "syndrome identification with medicine" is applied to study the TCM syndrome characters of the model and the action mechanism of intervention treatment for the model.METHODS:selected SD rats in child-bearing period checked with colpocytogram had normal estrous cycles and were included for study. Six of them were randomly selected as normal controls, and the others were all given intragastric administration with tripterygium glycosides 40mg/kg/day over 10 weeks to make the required models by using methods of our previous research. The models were successfully made when their monitored estrous cycles all became disordered.30 of the modeled rats were selected and randomly divided into five groups. Each group had six rats. Premarin and three different dosages (high, medium and low) of herbal compound "Tong Mai Da Sheng Pian" were given to four of the five groups respectively for 7 days, while one group set as normal model control was given similar volum of normal sodium for 7 days. Their general condition and representation were all noted. Their ovarian indexes were measured by tissue-weight-assay when sacrificed at the end of the treatment. Their serum "E2" and "P" were tested with LEIA, Inhibin B with ELISA, and "ER", " PR", "TNF-α/TNFR-1", " Caspase-3", "VEGF/Flk-1", and "Endostatin" with EIHCT. Pathological examinations were performed on their ovarian slides with HE stain to observe the histomorphological changes of ovarian cells with optical microscope.RESULTS:(1) Intragastric administration with tripterygium glycosides 40mg/kg/day over 10 weeks for a rat in child-bearing period can change it into a status of ovarian hypofunction which manifestations of bad general conditions and disordered estrous cycles, and significant decreases in their ovarian indexes, serum levels of E2, INHB growing follicale number, ER expression in ovarian tissue and VEGF expression in ovarian vessel, but obvious increase in the expressions of Endostatin, TNF-a and Caspase-3. The number of growing follicle in ovarian tissue decrease, and vessel wall thickening, lumen of blood vessel narrowing(2) After the interventional treatment with "Tong Mai Da Sheng Pian", the modeled rats can have obvious improvement in their general conditions with normal estrous cycles and enhanced ovarian indexes, and significant increases in E2, INHB, but obvious decreases in Endostatin, TNF-a and Caspase-3. The number of growing follicle in ovarian tissue increase, and vessel wall thinningz, lumen of blood vessel broaden.CONCLUSION:(1) The tripterygium glycosides can cause ovarian hypofunction in rats, while herbal compound "Tong Mai Da Sheng Pian" with functions to tonify Kidney Qi and nourish Blood and Essence can increase estrogen and ER levels, ovarian hemoperfusion, and inhibit ovarian apoptosis, and improve ovarian function effectively. (2) The effectiveness of treatment with the herbal compound tonifying Kidney verifies the rat model with TCM syndrome of Kidney Deficiency in Qi and Essence.更多还原