sRAGE对PCOS卵巢颗粒细胞AKT磷酸化及SP1表达的调控研究

发布时间：2018-05-19 11:11

本文选题：多囊卵巢综合征 + 可溶性晚期糖基化终产物受体　；参考：《郑州大学》2017年硕士论文

【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(P0.05);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(P0.05)。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(P0.05);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(P0.05)。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(P0.05)。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(P0.05)。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。
[Abstract]:Polycystic ovary syndrome (PCOS) is a systemic endocrine and metabolic disorder that occurs in women of childbearing age of 5-10%. Insulin resistance, Kaohsiung's steroid and follicular dysplasia are its main pathophysiological changes..PCOS can lead to infertility, internal secretion disorder, and so on. The long-term complications of cardiovascular disease, diabetes, and diabetes mellitus The incidence of disease, hypertension and so on is also increased. The pathogenesis of the quality of life of women and the health of.PCOS is not clear. The study found that inflammation, genetic factors, oxidative stress, environmental factors, social psychological factors and so on are all involved in the development of PCOS. The soluble advanced glycosylation end product receptor (soluble receptor for advanced) Glycation end-products, sRAGE) is an endogenous secretory type of RAGE, which inhibits multiple cell signaling pathways induced by AGE-RAGE axis by competing with RAGE in competition with RAGE. Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) can directly regulate oocyte maturation and early embryonic development, and can also regulate the follicular membrane. The microvascular network of the layer is formed to increase the vascular permeability to regulate the development of follicle. The serum VEGF level of.PCOS patients is significantly higher than that of non PCOS patients with the development of PCOS. The previous study in our group found that in PCOS ovarian granulosa cells, s RAEG is a protective factor, which can slow down the expression of VEGF and delay the occurrence and development of PCOS.V. The expression of EGF protein and m RNA decreases with the increase of sRAGE concentration, but the specific way sRAGE is to downregulate the expression of VEGF needs to further study the.PI3K/AKT pathway as a classic signaling pathway involved in the regulation of various cell metabolism. The central link is the activation of the.PI3K/AKT pathway of AKT, which activates a variety of SP transcription factors. SP1 is the main transcription factor regulating VEGF expression, and its binding with VEGF promoter plays a leading role in regulating VEGF transcription. In U937 derived foam cells, CD147 can up regulate the expression of VEGF through the PI3K/AKT pathway. In human ovarian epithelial cancer cells, resistin can enhance SP1 and VEGF promoter through the PI3K/AKT pathway. DNA binding activity up-regulated the expression of VEGF. Then, in PCOS ovarian granulosa cells, can sRAGE regulate the expression of SP1 by regulating AKT phosphorylation and then regulating the expression of VEGF? No related reports have been reported. This study aims to explore the regulatory role of sRAGE on PCOS ovarian granular cell AKT phosphorylation and SP1 expression, in order to further study sR. The basis of AGE action mechanism. Objective 1. to study the effect of sRAGE on the AKT phosphorylation of PCOS ovarian granulosa cells.2. to study the regulatory role of sRAGE on the expression of SP1 in PCOS ovarian granular cell transcription factors. Materials and methods were collected at the center of reproductive medicine in the 1st Affiliated Hospital of Zhengzhou University for 10 cases of follicle fluid in PCOS patients with IVF assisted pregnancy treatment, and the extraction of granular cells was obtained. After the 48h was cultured in vitro, the concentration was 0 mu g/ml, 0.6 g/ml and 1.2 g/ml sRAGE respectively. After adding 12h and 24h, the expression of AKT, P AKT, SP1 were detected respectively, and the protein expression was detected by Western blot. The effect of RAGE on the expression of AKT phosphorylation and transcription factor SP1 in PCOS ovarian granulosa cells. Results 1 mu g/ml, 0.6 mu g/ml, 1.2 micron g/ml sRAGE interfered with 12h and 24h of PCOS granular cells, and the relative expression of AKT protein in the three groups was not significantly different. The difference was statistically significant (P0.05).2.0 mu g/ml, 0.6 mu g/ml, and 1.2 mu g/ml sRAGE to treat PCOS granulosa cells 12h, and the expression of SP1 protein in 1.2 mu g/ml group was significantly lower than that of 0 mu g/ml group (P0.05). RAGE treatment of PCOS granulosa cells 12h, three groups of SP1m RNA expression decreased with the increase of sRAGE concentration, but the difference was not statistically significant (P0.05). After the treatment of 24h, the expression level of SP1m RNA decreased significantly with the increase of sRAGE concentration, the difference was statistically significant (P0.05). Moreover, a dose and time dependent.2.sRAGE can downregulate the expression of transcription factor SP1 in PCOS granulosa cells, which is dose and time dependent.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.75

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