【作者】 王晓丹；

【导师】 郭艺红；

【作者基本信息】 郑州大学 ， 生殖医学， 2017， 硕士

【摘要】 多囊卵巢综合征(polycystic ovary syndrome,PCOS)是一种全身性内分泌代谢紊乱疾病,发生于5-10%育龄期女性。胰岛素抵抗、高雄激素血症和卵泡发育异常是其主要的病理生理变化。PCOS会导致不孕、内分泌紊乱等,其远期并发症心血管疾病、糖尿病、高血压等的发病率也会增加,严重影响女性的生活质量及身体健康。PCOS的发病机制尚不明确,研究发现炎症、遗传因素、氧化应激、环境因素、社会心理因素等都参与PCOS的发生发展。可溶性晚期糖基化终产物受体(soluble receptor for advanced glycation end-products,sRAGE)是RAGE的内源分泌型,通过与RAGE竞争结合AGEs,从而抑制AGE-RAGE轴诱导的多种细胞信号传导途径。血管内皮生长因子(vascular endothelial growth factor,VEGF)能直接调控卵母细胞成熟和胚胎早期发育,也能够通过调控卵泡膜层的微血管网形成、增加血管通透性来调控卵泡发育,与PCOS的发生发展密切相关。PCOS患者血清VEGF水平明显高于非PCOS患者。本课题组前期研究发现,在PCOS卵巢颗粒细胞中,s RAEG作为保护性因子,能通过下调VEGF的表达,延缓PCOS的发生发展。VEGF蛋白和m RNA的表达量随sRAGE浓度的增加而降低。但sRAGE具体通过何种途径下调VEGF表达,尚需更深入的研究。PI3K/AKT通路是参与多种细胞代谢调节的经典信号通路,其中心环节为AKT的磷酸化。PI3K/AKT通路可激活包括SP转录因子在内的多种细胞因子。其中SP1是调控VEGF表达的主要转录因子,其与VEGF启动子的结合在调控VEGF转录中占据主导作用。在U937源性泡沫细胞中,CD147可通过PI3K/AKT通路上调VEGF的表达。在人卵巢上皮癌细胞中,抵抗素能通过PI3K/AKT通路增强SP1与VEGF启动子的DNA结合活性,上调VEGF的表达。那么,在PCOS卵巢颗粒细胞中,sRAGE能否通过调控AKT磷酸化影响转录因子SP1的表达进而调控VEGF的表达?目前未见相关报道。本研究旨在探究sRAGE对PCOS卵巢颗粒细胞AKT磷酸化及SP1表达的调控作用,为深入研究sRAGE作用机制奠定基础。目的1.研究sRAGE对PCOS卵巢颗粒细胞AKT磷酸化的调控作用。2.研究sRAGE对PCOS卵巢颗粒细胞转录因子SP1表达的调控作用。材料和方法收集于郑大一附院生殖医学中心进行IVF助孕治疗PCOS患者卵泡液共10例,提取颗粒细胞体外培养48h后分别添加浓度为0μg/ml,0.6μg/ml,1.2μg/ml的sRAGE进行干预。在添加后12h和24h分别检测AKT、p AKT、SP1的表达,通过蛋白质印迹法(Western blot)检测AKT、p AKT、SP1的蛋白表达,通过实时荧光定量聚合酶链反应(RT-q PCR)检测SP1的m RNA表达。探究sRAGE在PCOS卵巢颗粒细胞中对AKT磷酸化及转录因子SP1表达的调控作用。结果1.0μg/ml,0.6μg/ml,1.2μg/ml sRAGE分别干预PCOS颗粒细胞12h及24h,三组间AKT蛋白相对表达量均无明显统计学差异(P>0.05);而p AKT蛋白相对表达量随sRAGE浓度的增加而显著降低,差异有统计学意义(P<0.05)。2.0μg/ml,0.6μg/ml,1.2μg/ml sRAGE处理PCOS颗粒细胞12h,SP1蛋白表达量在1.2μg/ml组明显低于0μg/ml组(P<0.05);处理24h后检测,SP1蛋白表达量随sRAGE浓度的增加而显著降低,差异有统计学意义(P<0.05)。3.0μg/ml,0.6μg/ml,1.2μg/ml sRAGE处理PCOS颗粒细胞12h,三组SP1m RNA表达量随sRAGE浓度的增加有下降趋势,但差异无统计学意义(P>0.05)。处理24h后检测,SP1m RNA表达水平随sRAGE浓度的增加而显著降低,差异有统计学意义(P<0.05)。结论1.sRAGE可降低PCOS卵巢颗粒细胞AKT磷酸化水平,且具有剂量和时间依赖性。2.sRAGE可下调PCOS卵巢颗粒细胞中转录因子SP1的表达,且具有剂量和时间依赖性。更多还原

【Abstract】 Polycystic ovary syndrome(PCOS)is a systemic endocrine metabolic disorder,which affects almost 5-10% of women in reproductive age.The main pathophysiological changes of PCOS are insulin resistance,hyperandrogenemia and follicular dysplasia.PCOS can lead to infertility,and endocrine disorders.And the incidence of its long-term complications such as cardiovascular disease,diabetes and hypertension are increasing.Therefore,PCOS seriously affects the quality of life and physical health of women.The pathogenesis of PCOS is unclear,previous researches suggested that inflammation,genetic factors,oxidative stress,environmental factors and social psychological factors are involved in the occurrence and development of PCOS.Soluble receptor for advanced glycation end-products(sRAGE)is the endogenous secretory form of RAGE.sRAGE can inhibit multiple signal transduction pathways induced by AGE-RAGE axis by competing with RAGE.Vascular endothelial growth factor(VEGF)can act directly on oocyte maturation and early embryo development.VEGF can also regulate follicular development by regulating the formation of microvascular in theca cell layer and increasing the vascular permeability.Therefore VEGF is closely related to the occurrence and development of PCOS.The level of serum VEGF in patients with PCOS was significantly higher than that in non PCOS patients.The previous studies of our group have found that sRAGE,as a protective factor,can prevent the occurrence and development of PCOS by down regulating the expression of VEGF in granular cells.The expression of VEGF protein and mRNA decreased with the increasing of sRAGE concentration.However,the specific mechanism of how sRAGE down regulate VEGF expression remains unclear.Further research is needed to discover the mechanism.PI3K/AKT pathway is a classical signaling pathway involved in the regulation of cell metabolism.AKT phosphorylation is the center of this pathway.The PI3K/AKT pathway activates a variety of cytokines,including SP transcription factors.SP1 is the main transcription factor that regulates the expression of VEGF,and its binding with VEGF promoter plays a dominant role in the regulation of VEGF transcription.In U937 derived foam cells,CD147 can up regulate the expression of VEGF through PI3K/AKT pathway.In human ovarian epithelial cancer cells,resistin can enhance DNA binding activity of SP1 and VEGF promoter through the PI3K/AKT pathway,and then increase the expression of VEGF.Therefore,whether sRAGE can regulate the expression of VEGF by regulating the expression of transcription factor SP1 through the AKT pathway in granulosa cells of PCOS? There were no available research currently.The aim of this study was to investigate the effect of sRAGE on AKT phosphorylation and SP1 expression in PCOS ovarian granulosa cells,and to lay the foundation for further study of sRAGE mechanism.Objective1.To investigate the effect of sRAGE on AKT phosphorylation in PCOS ovarian granulosa cells.2.To investigate the effect of sRAGE on transcription factor SP1 expression in PCOS ovarian granulosa cells.Material and Methods1.Follicular fluid from 10 PCOS patients whom were treated with in vitro fertilization(IVF)in the reproductive medicine center of the First Affiliated Hospital of Zhengzhou University were collected.The granulosa cells were collected and cultured in vitro for 48 hours,and then treated with sRAGE at concentrations of 0 μg/ml,0.6 μg/ml and 1.2 μg/ml respectively.The expression of AKT,pAKT and SP1 were detected after adding sRAGE 12 h and 24 h respectively.The protein expression of AKT,pAKT and SP1 was detected by Western blotting.The mRNA expression of SP1 was detected by real-time quantitative polymerase chain reaction(RT-qPCR).To investigate the effect of sRAGE on AKT phosphorylation and transcription factor SP1 expression in PCOS ovarian granulosa cells.Results1.After treated with sRAGE at concentrations of 0 μg/ml,0.6 μg/ml and 1.2 μg/ml for 12 and 24 hours,there were no significant difference in the expression of AKT protein among three groups(P>0.05).While the expression of pAKT protein were significantly decreased with the increasing of sRAGE concentration,the differences were statistically significant(P<0.05).2.After treated with sRAGE at concentrations of 0 μg/ml,0.6 μg/ml and 1.2 μg/ml for 12 hours,the expression of SP1 protein in 1.2 μg/ml group was significantly lower than that of 0 μg/ml group(P<0.05).After treatment for 24 hours,the expression of SP1 protein significantly decreased with the increasing of sRAGE concentration,the differences were statistically significant(P<0.05).3.After treated with sRAGE at concentrations of 0 μg/ml,0.6 μg/ml and 1.2 μg/ml for 12 hours,the expression of SP1 mRNA in the three groups decreased with the increase of sRAGE concentration,but the difference was not statistically significant(P>0.05).After treatment for 24 hours,the expression level of SP1 mRNA significantly decreased with the increase of sRAGE concentration,the differences were statistically significant(P<0.05).Conclusions1.sRAGE can decrease the level of AKT phosphorylation in PCOS granulosa cells with dose and time dependence.2.sRAGE can down regulate the expression of transcription factors SP1 in PCOS granulosa cells with dose and time dependence.更多还原