高水平胰岛素对早孕小鼠卵巢凋亡的调控

发布时间：2018-05-04 05:10

本文选题：胰岛素 + 凋亡　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:代谢综合征已成为世界范围内的健康问题。以高血糖、肥胖、高血压和胰岛素抵抗为特征的代谢综合征及其合并症,对人群健康构成严重威胁。胰岛素抵抗及其相关的代谢综合征,与育龄期女性的生殖问题密切相关。子宫内膜蜕膜化对维持胚胎的正常发育具有重要作用。子宫内膜蜕膜化的异常可导致胚胎无法正常获取营养,从而致胚胎不能正常发育。课题组在前期研究中已经证实高水平胰岛素对早孕小鼠子宫内膜蜕膜化有影响,但其机制尚不明确。卵巢分泌的雌激素和孕酮对子宫内膜蜕膜化至关重要。有研究已经证实胰岛素可通过一系列信号通路调节细胞凋亡,且卵巢细胞的凋亡平衡是蜕膜化正常进行的重要保证。因此,本文旨在研究高水平胰岛素对妊娠早期卵巢细胞凋亡的影响,并进一步探讨其可能的机制。方法:1.动物模型的建立及标本收集:购买8周龄昆明鼠饲养,分别建立正常组和高胰岛素小鼠模型。将正常(或结扎输精管)雄鼠与雌鼠进行交配,次日见阴栓标记为D1(或PD1),分别收集正常组与高胰岛素组D7、D8(或PD7、PD8)的血清和卵巢组织。2.高胰岛素对早孕小鼠卵巢凋亡的影响:(1)TUNEL方法检测早孕小鼠D8卵巢细胞的调亡;(2)免疫组化检测孕D8小鼠卵巢Bax和Bcl2的表达;(3)westernblot检测正常妊娠(d7,d8)以及假孕(pd7,pd8)小鼠卵巢凋亡相关蛋白的表达。3.高浓度胰岛素对早孕小鼠卵巢pi3k/akt信号通路相关蛋白的影响:(1)westernblot检测正常妊娠(d7,d8)以及假孕(pd7,pd8)小鼠卵巢pi3k/akt信号通路相关蛋白的表达;(2)宫角注射pi3k/akt信号通路特异抑制剂ly294002,进一步分析pi3k/akt信号通路是否参与高浓度胰岛素对早孕小鼠卵巢细胞凋亡的影响。结果:1.高浓度胰岛素对早孕小鼠激素分泌的影响:elisa结果显示高胰岛素组小鼠d7和d8天血清中胰岛素浓度显著升高,而e2、p4浓度均显著降低。2.高浓度胰岛素对早孕小鼠卵巢凋亡的影响:tunel结果显示高胰岛素组早孕小鼠卵巢细胞凋亡显著降低;免疫组化结果显示高胰岛素组早孕小鼠卵巢组织bax表达显著下降而bcl2表达显著升高;westernblot结果显示高胰岛素组早孕小鼠卵巢组织bax、cleaved-casepase3、cleaved-parp表达明显降低而bcl2表达明显升高。3.pi3k/akt信号通路参与高胰岛素抑制的早孕小鼠卵巢细胞凋亡:westernblot结果显示akt磷酸化在高胰岛素组早孕小鼠卵巢组织中被激活。抑制剂ly294002能够改善高水平胰岛素引起的卵巢凋亡以及pi3k/akt信号通路相关蛋白的表达。结论:高胰岛素可能抑制早孕小鼠卵巢组织的凋亡,pi3k/akt信号通路可能参与这一过程,但其具体机制还有待进一步研究。
[Abstract]:Objective: metabolic syndrome has become a worldwide health problem. Metabolic syndrome and its complication, characterized by hyperglycemia, obesity, hypertension and insulin resistance, constitute a serious threat to the health of the population. Insulin resistance and its associated metabolic syndrome are closely related to reproductive problems in women of childbearing age. Endometrial deciduization The abnormal decidua of the endometrium can lead to the failure of the embryo to get the normal nutrition, so that the embryo can not develop normally. In the previous study, the research group has confirmed that the high level insulin has a shadow on the endometriosis of the endometrium in the early pregnancy mice, but the mechanism is not clear. The ovary secreted by the ovary is not clear. Hormone and progesterone are essential for endometrial decidualization. Studies have shown that insulin can regulate apoptosis through a series of signaling pathways, and the apoptosis balance of ovarian cells is an important guarantee for normal decidualization. Therefore, this article aims to study the effect of high level of insulin on the apoptosis of ovarian cells in early pregnancy and to further study the effect of high level insulin on the early ovarian cell apoptosis. To explore the possible mechanism. Methods: 1. animal model establishment and specimen collection: 8 weeks old Kunming mice were bought to establish normal group and high insulin mouse model. Normal (or ligated vasa) male rats were copulated with female rats. The second day was D1 (or PD1), and the normal group and the high insulin group were collected, D7, D8 (or PD7, PD8), respectively. The effect of.2. hyperinsulinemia in serum and ovarian tissue on ovarian apoptosis in early pregnancy mice: (1) TUNEL method was used to detect the apoptosis of D8 ovarian cells in early pregnant mice; (2) immunohistochemistry was used to detect the expression of Bax and Bcl2 in the ovary of pregnant D8 mice; (3) Westernblot detection of normal pregnancy (D7, D8) and the expression of apoptosis related proteins in the ovary of mice (pd7, pd8) were highly concentrated The effect of insulin on the pi3k/akt signaling pathway related proteins in the ovary of early pregnancy mice: (1) Westernblot detection of normal pregnancy (D7, D8) and the expression of pi3k/akt signal pathway related proteins in the ovary of mice (pd7, pd8); (2) a specific inhibitor of pi3k/akt signaling in the uterine horn, LY294002, to further analyze the involvement of pi3k/akt signaling pathway. Effect of insulin concentration on the apoptosis of ovarian cells in early pregnancy mice. Results: 1. the effect of high insulin on the hormone secretion in early pregnancy mice: ELISA results showed that the serum insulin concentration in D7 and D8 days in high insulin group increased significantly, while E2, P4 concentration significantly reduced the effect of.2. high concentration insulin on premature ovarian apoptosis in mice: tunel The results showed that the apoptosis of ovarian cells in the early pregnancy mice was significantly lower than that in the high insulin group, and the immunohistochemical results showed that the expression of Bax in the ovarian tissue of the early pregnant mice was significantly decreased while the expression of BCL2 in the early pregnancy mice increased significantly, and the results of Westernblot showed that the expression of Bax, cleaved-casepase3 and cleaved-parp in the early pregnancy mice of the hyperinsulinic group was significantly reduced by BC. L2 expression significantly increased.3.pi3k/akt signaling pathway involved in the apoptosis of ovarian cells in early pregnancy mice with high insulin inhibition. Westernblot results showed that Akt phosphorylation was activated in the ovarian tissue of early pregnant mice with high insulin. Inhibitor LY294002 could improve the ovarian apoptosis induced by high level insulin and pi3k/akt signaling pathway related proteins. Conclusion: high insulin may inhibit the apoptosis of ovarian tissue in early pregnancy mice. Pi3k/akt signaling pathway may be involved in this process, but its specific mechanism remains to be further studied.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714

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