【作者】 王芳；

【导师】 张治芬；

【作者基本信息】 南京医科大学 ， 妇产科（专业学位）， 2019， 博士

【摘要】 研究背景:多囊卵巢综合征(polycystic ovary syndrome,PCOS)病因复杂,症状多样,是引起育龄期女性不孕不育的主要疾病之一,主要表现为稀发排卵/无排卵、糖脂及性激素代谢紊乱。其中卵巢间质纤维化、包膜增厚、卵泡发育异常是引起排卵障碍的主要病理改变,其发生机制尚不清楚。研究现状:以往研究表明以转化生长因子β1(Transforming growth factor-β1,TGF-β1)为中心的信号通路可能参与PCOS的多种病理改变。遗传学研究发现,TGF-β1基因的某些核苷酸位点多态性与PCOS发生相关。尚无证据表明TGF-β1通路及相关分子参与PCOS卵巢纤维化进程,亦缺少DPP4抑制剂对TGF-β1通路在PCOS中影响的报道。研究目的:1.通过分析TGF-β1信号通路对PCOS大鼠生殖、代谢以及卵巢病理等方面的影响,探讨TGF-β1信号通路在PCOS中的转导调控机制;2.通过检测人群中TGF-β1基因rs4803457基因及基因型频率,探讨TGF-β1 rs4803457单核苷酸多态性(Single Nucleotide Polymorphism,SNP)对PCOS的影响。研究方法:1.将30只雌性大鼠随机分为对照组、PCOS模型组和西格列汀治疗组,观察卵巢细胞形态和间质内胶原纤维比例;测定血中性激素抗缪勒管激素(Anti-Mullerian Hormone,AMH)、黄体生成素(Luteinizing hormone,LH)、睾酮(Testosterone,T)、卵泡生成激素(follicle-stimulating hormone,FSH)和血糖血脂水平包括血糖(Glucose,Glu)、胰岛素(Insulin,INS)、低密度脂蛋白(Low density lipoprotein,LDL)、高密度脂蛋白(High density lipoprotein,HDL)、甘油三酯(Triglyceride,TG)、总胆固醇(Total Cholesterol,TC);检测卵巢内二肽基肽酶IV(Dipeptidyl peptidase-4,DPP4)、TGF-β1、Smad2/3、结缔组织生长因子(Connection tissue growth factor,CTGF)蛋白及相应mRNA在卵巢中的表达及含量。2.选取PCOS患者205例和健康女性207例,测定DNA中TGF-β1基因rs4803457位点的基因型频率和等位基因频率,检测生化指标(Glu、TC、INS)及性激素(T、LH、FSH)。研究结果:1.大鼠模型组卵巢结构紊乱,呈典型多囊样改变,治疗组卵巢结构较规整。III型胶原在各组卵巢中所占比例均高于I型,模型组III型及I型胶原纤维百分比均明显高于对照组及治疗组。2.DPP4、TGF-β1、Smad2/3、CTGF蛋白可表达于大鼠卵巢间质、卵泡膜细胞及颗粒细胞,以卵巢间质为主,模型组尤其明显;DPP4、TGF-β1、CTGF mRNA水平在模型组均明显高于对照组和治疗组;从卵巢整体看来三组间Smad2/3蛋白及mRNA水平差异无显著性,但卵巢间质中,模型组Smad2/3蛋白表达均明显增强;治疗组Smad3表达强度明显减弱。3.三组大鼠间TC、TG、LDL、LH/FSH比较,无明显差异。模型组T、AMH较对照组偏高,HDL低于对照组。治疗组Glu、INS、HOMA、T较模型组降低,HDL高于模型组。4.人群中PCOS组肥胖与非肥胖患者比较,TGF-β1 rs4803457CC基因型OR为1.903,95%CI(1.025-3.530)。结论和展望:TGF-β1通过TGF-β1/Smad2/3/CTGF信号通路参与PCOS卵巢病理学进展,DPP4抑制剂可以阻断该通路,从而起到逆转卵巢纤维化、促进排卵作用,同时发挥减轻胰岛抵抗、调节性激素的作用;TGF-β1基因rs4803457单核苷酸多态性与PCOS相关,CC基因型是PCOS肥胖的危险因素。更多还原

【Abstract】 Background: Polycystic ovary syndrome(PCOS)causes complex and diverse symptoms.It is one of the main diseases causing infertility in women of childbearing age,mainly characterized by sparse ovulation or disturbance of glycolipid and sex hormone metabolism.Ovarian stroma fibrosis,thickening of the capsule,and abnormal follicular dysplasia are the main pathological changes that cause ovulation disorders,and the mechanism is still unclear.Current status of research: Previous studies have shown that Transforming growth factor-β1(TGF-β1)signaling pathway might be involved in various pathological changes of PCOS.Genetic studies have found that certain nucleotide polymorphisms in the TGF-β1 gene are associated with risk of PCOS.There is no evidence that TGF-β1 signaling pathway and related molecules are involved in PCOS ovarian fibrosis yet.Current evidence is not enough to prove that DPP4 inhibitor effects TGF-β1pathway in PCOS.Objectives: 1.To investigate the mechanism of regulation on TGF-β1 signaling pathway for PCOS by analyzing the effects of TGF-β1 signaling pathway on reproductive,metabolic and pathological changes in PCOS rats;2.To investigate the effects of TGF-β1 single nucleotide polymorphism(SNP)on PCOS by analyzing the genotype frequency and allele frequency of TGF-β1 gene rs4803457.Methods: 1.30 female rats were divided into blank control group,PCOS model group and Siglitin treatment group.After execution,the ovaries were analysed including the cell morphology and the proportion of collagen fibers in the interstitium.the serum of sex hormones,blood glucose and lipid were detected,including anti-Müllerian hormone(AMH),Luteinizing hormone(LH),Testosterone(T),follicle-stimulating hormone(FSH),Glucose(Glu),Insulin(INS)、Low density lipoprotein(LDL),High density lipoprotein(HDL),Triglyceride(TG)、Total Cholesterol(TC).The protein and mRNA expression of Dipeptidyl peptidase-4(DPP4),TGF-β1,Smad2/3 and Connection tissue growth factor(CTGF)in different ovarian cells were detected too.2.205 PCOS women and 207 healthy controls were selected.The genotype frequency and allele frequency of TGF-β1 gene rs4803457 in DNA were determined.Biochemical indicators(Glu,TC,INS)and sex hormones(T,LH,FSH)were detected.Results: 1.Structural disorder of ovarian were observed in rat model group.The tissue appeared typical polycystic changes.while the ovarian structure in treatment group is more regular.The proportion of type III collagen in each group of ovaries was higher than that of type I.The percentage of type III and type I collagen fibers in the model group was significantly higher than that in the control group and the treatment group.2.DPP4,TGF-β1,Smad2/3,CTGF protein can be expressed in rat ovarian stroma,ovarian membrane and granulosa cells,mainly in ovarian stroma,especially in model group;DPP4,TGF-β1,CTGF mRNA level in the model group was significantly higher than that in the control group and the treatment group.For the whole ovary,there was no significant difference in Smad2/3 protein and mRNA levels.However,in the ovarian stroma,the expression of Smad2/3 in the model group was significantly increased.The intensity of Smad3 expression was significantly attenuated in treatment group.3.There were no significant differences in TC,TG,LDL and LH/FSH between the three groups.The levers of AMH and T in the model group were higher than the control group,and the HDL was lower than the control group.Glu,INS,HOMA,and T in the treatment group were lower than those in the model group,and HDL was higher than the model group.4.The OR of TGF-β1 rs4803457 CC genotype was 1.903 and 95% CI(1.025-3.530)in obesity and non-obesity patients in PCOS group.Conclusions and prospects: The results suggest that TGF-β1 participates in the progress of PCOS ovarian pathology through TGF-β1/Smad2/3/CTGF signaling pathway,and DPP4 inhibitors can block this pathway,thereby reversing ovarian fibrosis and promoting ovulation,alleviate islet resistance and regulate the role of sex hormones.The single nucleotide polymorphism of TGF-β1 gene rs4803457 is associated with PCOS,and CC genotype may be a risk factor for PCOS obesity.更多还原