Smad2、Smad3及Smad7蛋白在绝经过渡期大鼠卵巢中的表达及意义

发布时间：2018-05-07 18:21

本文选题：卵巢功能衰退 + POF　；参考：《河北北方学院》2017年硕士论文

【摘要】：卵巢功能衰退是指由于女性卵巢的生殖功能和内分泌功能下降导致月经紊乱、生育力下降及低雌激素症状的发生,也可称为卵巢储备功能下降(Decreasing Ovarian Reservation,DOR),包括围绝经期卵巢功能衰退和卵巢早衰(premature ovarian failure,POF)。卵巢功能衰退意味着女性开始进入衰老,严重影响着女性生殖健康及预期健康生命质量。因此探寻卵巢衰老中内在分子机制,寻找靶向目的蛋白改善卵巢功能从而延缓卵巢功能衰退具有重大意义。卵巢功能衰退存在极大的个体差异性,提示卵巢内微环境起着重要作用,本课题以绝经过渡期雌性大鼠为研究对象,着眼于与卵巢功能密切相关的转化生长因子-β(trans-forming growth factorβ,TGF-β)信号通路中的细胞内信号转导蛋白Smad蛋白,探究在卵巢功能衰退中受体激活型Smad蛋白Smad2、Smad3及抑制型Smad蛋白Smad7的蛋白水平及基因水平的表达变化,初步研究Smad蛋白与卵巢功能衰退的关系。本课题选取阴道涂片筛选法验证的12月龄绝经过渡期雌性SD(Sprague-Dawley)大鼠28只,,随机分成绝经过渡期组(MT)、雌二醇治疗绝经过渡期组(E2),每组14只,另选14只6月龄雌性大鼠作为性成熟对照组(C)。E2治疗组皮下注射17-β雌二醇(0.2mg/kg,1次/3天),MT组及C组皮下注射同等剂量的芝麻油溶剂6周后取双侧卵巢,计算卵巢指数、行HE染色观察卵巢形态和卵泡数量计数评估各组卵巢功能,通过实时荧光定量PCR法检测Smad2,Smad3,Smad7基因在各组卵巢组织中的mRNA表达差异,免疫组织化学方法检测Smad2、P-Smad2、Smad3、P-Smad3、Smad7蛋白的在各组卵巢中的定位和表达变化。根据卵巢指数及形态学计算各级卵泡数量结果发现MT组及E2组卵巢功能较C组降低,MT组卵巢功能为最低,结果有统计学意义(P0.05)。RT-q PCR结果显示MT组、E2组Smad2 mRNA、Smad3mRNA表达水平显著低于C组(P0.01),E2组Smad2、Smad3 mRNA水平显著高于MT组(P0.05);Smad7 mRNA水平在MT组、E2组中显著高于C组(P0.01),E2组低于MT组(P0.05)。免疫组织化学结果表明Smad2、Smad3、P-Smad2、P-Smad3及Smad7蛋白在颗粒细胞、卵母细胞及卵泡膜细胞中均有表达,三组间有表达差异。Smad2,Smad3蛋白水平在C组、E2组、MT组中呈逐渐下降趋势,具有统计学意义(P0.05),相同P-Smad2、P-Smad3蛋白水平呈相同趋势,结果无统计学意义;而Smad7蛋白水平在C组、E2组、MT组呈升高趋势,具有统计学意义(P0.05)。通过对以上结果分析得出,Smad2、Smad3、Smad7在卵巢卵泡细胞中广泛表达,对卵巢功能起调控作用。在卵巢功能衰退过程中,无论从蛋白水平及基因水平,均有Smad2,Smad3,Smad7参与,Smad2、Smad3起正向调控,Smad7起负向调控作用。卵巢功能衰退与Smad2,Smad3表达下调,Smad7表达上调有关。
[Abstract]:The decline of ovarian function refers to the disorder of menstruation, fertility decline and the occurrence of low estrogen symptoms due to the decline of reproductive and endocrine functions of female ovaries. Also known as ovarian reserve decline, decreasing Ovarian reservation, including peri-menopausal ovarian dysfunction and premature ovarian failure. The decline of ovarian function means that women begin to age, which seriously affects their reproductive health and the quality of life of their expected health. Therefore, it is of great significance to explore the intrinsic molecular mechanism of ovarian senescence and to find target protein to improve ovarian function and delay ovarian function decline. There are great individual differences in ovarian dysfunction, suggesting that the microenvironment plays an important role in the study of menopausal transition female rats. We focus on the intracellular signal transduction protein Smad protein in transforming growth factor 尾 TGF- 尾 signaling pathway, which is closely related to ovarian function. To investigate the expression of receptor activated Smad protein Smad2Smad3 and inhibitory Smad protein Smad7 in ovarian dysfunction, and to explore the relationship between Smad protein and ovarian dysfunction. In this study, 28 postmenopausal female SD Sprague-Dawley rats verified by vaginal smear screening were randomly divided into menopausal transition group (MTO) and estradiol group (14 rats in each group). Another 14 6-month-old female rats were selected as the control group of sexual maturation. The ovarias were obtained by subcutaneous injection of 17- 尾 estradiol 0.2mg / kg of estradiol 0.2mg / kg in group C and group C by subcutaneous injection of the same dose of sesame oil solvent for 6 weeks, and the ovarian index was calculated, and the ovarian index was calculated by subcutaneous injection of 17- 尾 estradiol 0.2mg / kg of estradiol. The morphology of ovary and the number of follicles were observed by HE staining to evaluate the ovarian function in each group. The difference of mRNA expression of Smad2 Smad3 Smad7 gene in ovarian tissues was detected by real-time fluorescence quantitative PCR method. Immunohistochemical method was used to detect the localization and expression of Smad2T P-Smad3P- Smad3 Smad7 protein in the ovaries of each group. According to the ovarian index and morphology, the ovarian function of MT and E2 group was lower than that of C group, and the ovarian function of MT group was the lowest. Results the results showed that the expression of Smad2 mRNA-Smad3 mRNA in MT group was significantly lower than that in group C (P 0.01). The level of Smad3 mRNA was significantly higher in MT group than that in MT group. The level of Smad7 mRNA in MT group was significantly higher than that in group C (P 0.01) and that in group C was significantly higher than that in group C (P 0.01). The results of immunohistochemistry showed that Smad2Smad3 and Smad7 protein were expressed in granulosa cells, oocytes and follicular cells, and the Smad2 Smad3 protein level decreased gradually in group C and E 2 group, and the expression of Smad3 protein was decreased gradually in group C and E 2 group, and the expression of Smad2 Smad3 protein was decreased gradually in group C and E _ 2 group, and the expression of Smad2 Smad3 protein was decreased gradually in E _ 2 group and T _ T group. The level of P-Smad2, P-Smad3 protein showed the same trend, but the level of Smad7 protein increased in group C (P 0.05). Through the analysis of the above results, it is concluded that Smad2, Smad3, Smad7 are widely expressed in ovarian follicular cells and play a regulatory role in ovarian function. In the course of ovarian function decline, Smad2, Smad3 and Smad7 play a negative role in the positive regulation of Smad7 both in protein level and gene level. The decline of ovarian function was associated with down-regulation of Smad3 expression and up-regulation of Smad7 expression.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.75

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