【摘要】：目的:建立慢性不可預(yù)見性溫和應(yīng)激小鼠模型;觀察慢性不可預(yù)見性溫和應(yīng)激對小鼠卵巢反應(yīng)性、卵母細胞發(fā)育潛能的影響,探討與卵母細胞發(fā)育潛能相關(guān)的局部調(diào)控因子BDNF是否參與了慢性不可預(yù)見性溫和應(yīng)激對小鼠卵巢反應(yīng)性和卵母細胞發(fā)育潛能的影響。 方法: (1)利用孤養(yǎng)、饑餓、冷水游泳等連續(xù)溫和應(yīng)激相繼施與小鼠30天,建立慢性不可預(yù)見性溫和應(yīng)激雌性小鼠模型,曠場試驗檢測行為學(xué)、ELISA方法檢測血清學(xué)皮質(zhì)酮、免疫組化方法檢測下丘腦CRH陽性神經(jīng)元變化鑒定慢性應(yīng)激動物模型。(2)觀察PMSG+HCG促排卵后的獲卵數(shù)、卵母細胞孤雌激活后體外培養(yǎng)至囊胚階段觀察慢性應(yīng)激對卵巢反應(yīng)性和卵母細胞發(fā)育潛能的影響。(3)透視電子顯微鏡觀察卵母細胞和顆粒細胞超微結(jié)構(gòu)、卵巢組織免疫組化方法檢測卵巢BDNF水平探討慢性應(yīng)激對小鼠卵巢卵巢反應(yīng)性和卵母細胞發(fā)育潛能可能的機制。 結(jié)果: (1)通過連續(xù)30天的慢性不可預(yù)見性溫和應(yīng)激后,行為學(xué)檢測顯示小鼠中央格停留時間延長、水平運動和直立運動明顯減少;血清學(xué)檢測顯示慢性應(yīng)激后小鼠皮質(zhì)酮水平明顯升高;免疫組化方法顯示慢性應(yīng)激后下丘腦室旁核CRH陽性神經(jīng)元增多。(2)慢性應(yīng)激后小鼠促排卵獲卵數(shù)明顯減少;卵母細胞孤雌激活后體外培養(yǎng),囊胚形成率顯著降低。(3)慢性應(yīng)激后透視電子顯微鏡下觀察小鼠卵巢超微結(jié)構(gòu)顯示卵母細胞與顆粒細胞間縫隙連接破壞、顆粒細胞凋亡增多;卵巢組織免疫組化結(jié)果顯示卵巢竇卵泡、成熟卵泡內(nèi)BDNF表達降低。 結(jié)論:慢性不可預(yù)見性溫和應(yīng)激后小鼠行為學(xué)呈抑郁樣改變、HPA軸活性升高,說明小鼠處于應(yīng)激狀態(tài);慢性應(yīng)激后小鼠獲卵數(shù)減少、囊胚形成率降低,提示慢性應(yīng)激降低了卵巢反應(yīng)性和卵母細胞發(fā)育潛能;慢性應(yīng)激小鼠卵泡內(nèi)顆粒細胞凋亡增加、卵母細胞與顆粒細胞間的縫隙連接破壞;慢性應(yīng)激可致竇卵泡和成熟卵泡內(nèi)BDNF表達水平下降。以上結(jié)果提示慢性應(yīng)激可能通過抑制卵巢內(nèi)自分泌和旁分泌因子BDNF的表達,進而抑制小鼠顆粒細胞生長和增殖,并抑制卵母細胞和顆粒細胞之間的縫隙連接交流,從而對卵泡的生長,卵母細胞的發(fā)育潛能產(chǎn)生影響。
[Abstract]:Objective: to establish a mouse model of chronic unpredictable mild stress. To observe the effect of chronic unpredictable mild stress on ovarian reactivity and oocyte development potential in mice. To investigate whether the local regulatory factor BDNF associated with oocyte development potential is involved in the effects of chronic unpredictable mild stress on ovarian reactivity and oocyte development potential in mice. Methods: (1) the female mice model of chronic unpredictable mild stress was established by continuous mild stress, such as solitary rearing, starvation and cold water swimming for 30 days. Behavior was detected by open field test and serum corticosterone was detected by ELISA method. The changes of CRH positive neurons in hypothalamus were detected by immunohistochemical method to identify the chronic stress animal model. (2) the number of eggs obtained after ovulation induced by PMSG HCG was observed. The effects of chronic stress on ovarian reactivity and oocyte development potential were observed in vitro after parthenogenetic activation. (3) the ultrastructure of oocytes and granulosa cells were observed by fluoroscopy. Detection of ovarian BDNF level by immunohistochemical method in ovarian tissues to explore the possible mechanism of chronic stress on ovarian responsiveness and oocyte development potential in mice. Results: (1) after 30 days of chronic unpredictable mild stress, the behavior test showed that the retention time of central lattice was prolonged, the horizontal movement and upright movement were decreased obviously. The serum level of corticosterone was significantly increased after chronic stress, the number of CRH positive neurons in paraventricular nucleus of hypothalamus was increased after chronic stress. (2) the number of ovulation induction was significantly decreased after chronic stress. The blastocyst formation rate was significantly decreased after parthenogenetic activation of oocytes in vitro. (3) the ultrastructure of mouse ovary after chronic stress showed that the gap junctions between oocytes and granulosa cells were damaged. Granulosa cell apoptosis increased; The expression of BDNF in ovarian antral follicles was lower than that in mature follicles. Conclusion: after chronic unpredictable mild stress, the behavior of mice showed depressive changes and the activity of HPA axis increased, indicating that the mice were in stress state. The number of oocytes was decreased and the blastocyst formation rate was decreased after chronic stress, indicating that chronic stress decreased ovarian reactivity and oocyte development potential. The apoptosis of granulosa cells in follicles increased and the gap junction between oocytes and granulosa cells was destroyed in chronic stress, and the expression of BDNF in antral follicles and mature follicles decreased after chronic stress. These results suggest that chronic stress may inhibit the growth and proliferation of mouse granulosa cells by inhibiting the expression of autocrine and paracrine factor BDNF, and inhibit the gap junction communication between oocytes and granulosa cells. Thus, the growth of follicles and the developmental potential of oocytes are affected.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R321

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