【作者】 李燕；

【导师】 张庆文；

【作者基本信息】 成都中医药大学 ， 中医妇科学， 2013， 博士

【摘要】 目的：运用脱氢表雄酮诱导SD大鼠多囊卵巢综合征模型,研究多囊卵巢模型大鼠基质金属蛋白酶及其组织抑制因子(MMPs-TIMPs)的平衡失调,并运用基于化痰化瘀法的中药复方三贝地甲煎来调控多囊卵巢模型大鼠MMPs-TIMPs的平衡表达,为化痰化瘀法治疗多囊卵巢综合征提供科学依据。方法：1.每日于模型组大鼠颈背部皮下注射DHEA溶液(6mg/0.2ml/100g),连续注射20天诱导SD大鼠建立多囊卵巢综合征模型,采用ELISA法检测空白组和模型组大鼠的生殖激素T、LH、FSH值,血糖及胰岛素值等,制作卵巢病理切片观测组织学指标,验证DHEA能建立PCOS模型,并采用QPCR法检测卵巢MMP-2mRNA、 MMP-9mRNA、TIMP-1mRNA、TIMP-2mRNA的2-△△CT值以及MMPs/TIMPs的比值,研究PCOS状态下大鼠卵巢MMPs-TIMPs的平衡表达。所有数据均应用SPSS18.0统计软件进行整理和分析。计量资料用均数±标准差(x±s)形式表示,两组之间比较采用独立样本t检验。2.应用DHEA制作PCOS模型后,将大鼠分为空白组、生理盐水组、达因组、二甲双胍组、中药三贝地甲煎高、中、低剂量组共7组,分别灌服相应药物,同时每日阴道脱落细胞学涂片,当模型组某组大鼠50%以上个体出现第1个正常排卵周期时,处死全体动物,采用ELISA法检测各组的生殖激素T、LH、FSH值,血糖及胰岛素值等,制作卵巢病理切片观测组织学指标,采用QPCR法检测卵巢MMP-2mRNA、MMP-9mRNA、TIMP-lmRNA、TIMP-2mRNA的2-△△CT值以及MMPs/TIMPs的比值,对比各组药物产生的不同效果,研究各组药物通过调整MMPs-TIMPs的平衡而治疗PCOS的机理,特别是化痰化瘀中药三贝地甲煎对PCOS大鼠MMPs-TIMPs平衡表达的调控作用。所有数据均应用SPSS18.0统计软件进行整理和分析。计量资料用均数±标准差(x±s)形式表示,多组比较采用单因素方差分析,两两比较采用LSD-t检验。结果：1.DHEA诱导的SD雌大鼠出现卵巢指数明显升高,卵巢形态学发现成熟卵泡减少、闭锁卵泡增多、囊状扩张卵泡增多,血清T值增高,LH值增高,空腹血糖、空腹胰岛素、胰岛素抵抗指数均较空白组显著升高,基本符合PCOS的病理特点,且卵巢MMPs/TMPs比值升高。2.基于化痰化瘀法的三贝地甲煎中药复方能够降低PCOS大鼠卵巢指数,改善PCOS大鼠的卵巢形态学变化,降低PCOS大鼠血T值,降低PCOS大鼠的空腹血糖、空腹胰岛素及胰岛素抵抗指数,降低PCOS大鼠升高的MMP9/TIMP1比值和MMP2/TIMP2比值,从而调节MMPs-TIMPs的平衡表达,综合作用优于阳性药达因-35和二甲双胍。结论：DHEA能诱导大鼠SD多囊卵巢综合征模型；多囊卵巢模型大鼠存在MMPs-TIMPs的平衡失调；基于化痰化瘀法的三贝地甲煎中药复方能调控多囊卵巢模型大鼠MMPs-TIMPs平衡表达。更多还原

【Abstract】 Objective:The dehydroepiandrosterone (DHEA) was used to induce the model of SD rats with polycystic ovary syndrome (PCOS), in order to research the matrix metalloproteinases of polycystic ovary rats and the imbalance of their tissue inhibitory factors (MMPs-TIMPs), then the Chinese medicine Sanbeidijiajian (Compound medicine of Rhizoma Sparganii and Fritillaria etc.) with dissipating phlegm and removing stasis efficacy was used to regulate the MMPs-TIMPs expression balance of polycystic ovary rats, so as to provide a scientific basis for the treatment of PCOS.Methods:1. The DHEA solution was administrated to the rats daily by subcutaneous injection (6mg/0.2ml/100g) for20consecutive days in order to induce the models of PCOS, and then the reproductive hormone T, LH, FSH values, blood glucose and insulin levels, ovarian histology indicators of both blank group and model group were determined by ELISA assay, the DEHA were verified to build PCOS models, next, the2-ΔΔCT values and the MMPs/TIMPs ratios of the ovarian MMP-2mRNA、MMP-9mRNA、 TIMP-1mRNA、TIMP-2mRNA were determined by QPCR assay, so as to study the expression balance of the of MMPs-TIMPs under PCOS condition. All data were collected and analyzed by using SPSS18.0statistical software. Measurement data were expressed as form of mean±standard deviation (x±s), and the data of both groups were compared independent samples Student’s t-test.2. After the application of DHEA for the formation of PCOS models, the rats were randomly divided into7groups including blank group, saline group, Diane group, metformin group, Chinese medicine Sanbeidijiajian high, middle, low dose groups and were orally administrated their corresponding medicines, and they were performed vaginal exfoliative cytology smear test daily, when50%of rats in one of model groups had their first normal ovulation cycles, all the animals were sacrificed. Then the reproductive hormone T, LH, FSH values, blood glucose and insulin levels, ovarian histology indicators of every group were determined by ELISA assay, and the2-ΔΔ CT values and the MMPs/TIMPs ratios of the ovarian MMP-2mRNA、 MMP-9mRNA、TIMP-1mRNA、TIMP-2mRNA were determined by QPCR assay, so as to compare different effects produced by the medicines in every group and explore the treatment mechanisms of these medicines to PCOS based on their regulations on the expression balance of MMPs-TIMPs, especially Chinese medicine Sanbeidijiajian. All data were collected and analyzed by using SPSS18.0statistical software. Measurement data were expressed as form of mean±standard deviation (x±s), the single-factor variance analysis was used for multi-group comparison, and the LSD-t test was used for the data comparisons between any two of all these groups.Results:DHEA-induced rats had significant high ovarian indexes, and ovarian morphologic changes such as decreased mature follicles, increased atretic follicles, increased follicular cystic dilatations, increased serum T levels and LH values, and higher fasting blood glucose, fasting insulin, insulin resistance indexes than those of blank group, as well as high MMPs/TIMPs ratio, which were basically in line with the pathological features of PCOS. The Chinese medicine Sanbeidijiajian with dissipating phlegm and removing stasis efficacy can effectively reduce the ovarian indexes of PCOS rats and improve their ovarian morphological changes, as well as decrease their serum T levels, fasting blood glucose, fasting insulin, insulin resistance indexes, decrease their abnormal MMP9/TIMP1ratio and MMP2/TIMP2ratio, thereby regulating the expression balance of MMPs-TIMPs to produce a comprehensive effect better than that of positive drug result Diane-35or metformin. This medicine has an excellent therapeutic effect for PCOS.Conclusion:DHEA can be used to induce the SD PCOS rats model; the PCOS rats have MMPs-TIMPs expression imbalance; the Chinese medicine Sanbeidijiajian with dissipating phlegm and removing stasis efficacy can effectively regulate the expression balance of MMPs-TIMPs in PCOS rats.更多还原