【摘要】：浙東白鵝是中國肉鵝中著名的優(yōu)良品種,具有生長速度快、肉質(zhì)鮮美等特點,然而其就巢性強,產(chǎn)蛋量低,使品種的發(fā)展收到嚴重阻礙。對就巢期的母鵝的卵巢進行觀察,發(fā)現(xiàn)其中卵泡均發(fā)生不同程度的閉鎖,眾多研究表明,禽類卵泡中顆粒細胞凋亡是導(dǎo)致其卵泡閉鎖的主要原因。哺乳動物中的研究顯示,細胞凋亡與細胞自噬密切相關(guān),且作用機制復(fù)雜。鑒于此,本試驗試圖從自噬的角度研究卵泡閉鎖的相關(guān)機制。試驗通過透射電鏡、免疫組化等方法,對正常生長卵泡和閉鎖卵泡中自噬體進行定位與觀察,并采用Western blot、熒光定量PCR等技術(shù),對自噬體標記蛋白LC3的表達量進行檢測,判斷卵泡閉鎖是否與細胞自噬相關(guān)。同時,通過多個生化指標的測定,檢測氧化應(yīng)激是否通過誘導(dǎo)卵泡細胞的自噬促進卵泡閉鎖。此外,在動物試驗結(jié)果的基礎(chǔ)上,通過培養(yǎng)原代鵝卵泡顆粒細胞,建立氧化應(yīng)激模型,以進一步在細胞水平進行驗證。在鵝卵泡顆粒細胞氧化模型的基礎(chǔ)上,分別加入Rapamycin和3-methyladenine(3-MA),以檢測卵巢顆粒細胞中mTOR信號通路和Beclin-1信號通路,探究氧化應(yīng)激誘導(dǎo)鵝卵泡顆粒細胞自噬的作用機制。研究結(jié)果表明:與生長卵泡相比,閉鎖卵泡顆粒細胞中自噬水平升高(P0.05),氧化應(yīng)激水平升高(P0.05)。通過H2O2誘導(dǎo)顆粒細胞建立氧化模型,檢測發(fā)現(xiàn)自噬水平顯著升高(P0.05)。對氧化應(yīng)激模型進行3-MA或Rapamycin共處理,發(fā)現(xiàn)Rapamycin共處理組LC3-Ⅱ蛋白表達量顯著升高(P0.05),自噬體數(shù)量顯著增多(P0.05),而3-MA對顆粒細胞自噬水平無影響。研究結(jié)論:本研究證明了鵝就巢期卵泡閉鎖與顆粒細胞自噬相關(guān),明確了氧化應(yīng)激引起的內(nèi)環(huán)境變化是誘導(dǎo)顆粒細胞自噬的重要因素。且在氧化條件下,mTOR信號通路是誘導(dǎo)顆粒細胞發(fā)生自噬的主要途徑。
[Abstract]:Zhejiang Eastern White Goose is a famous goose breed in China. It has the characteristics of fast growth and delicious meat. However, its strong nesting ability and low egg production have seriously hindered the development of the goose breed. Granulocyte apoptosis is the main cause of follicular atresia. Studies in mammals have shown that apoptosis is closely related to autophagy and the mechanism of action is complex. The expression of autophagy-labeled protein LC3 was detected by Western blot and fluorescence quantitative PCR to determine whether follicular atresia was associated with autophagy. At the same time, the effects of oxidative stress on follicular autophagy were detected by measuring several biochemical parameters. In addition, on the basis of animal experiments, the oxidative stress model was established by culturing primary follicular granulosa cells of goose to further validate at the cellular level. Rapamycin and 3-methyladenine (3-MA) were added to the oxidative model of follicular granulosa cells to detect the mTOR signaling pathway and Be in ovarian granulosa cells, respectively. Clin-1 signaling pathway was used to explore the mechanism of oxidative stress-induced autophagy in granulosa cells of goose follicles. The results showed that autophagy level in granulosa cells of atresia follicles increased (P 0.05) and oxidative stress level increased (P 0.05) compared with growth follicles. The expression of LC3-II protein was significantly increased (P 0.05) and the number of autophagy was significantly increased (P 0.05) in the Rapamycin co-treatment group, while 3-MA had no effect on the level of autophagy of granulosa cells. The change of internal environment induced by oxidative stress is an important factor in inducing autophagy of granulosa cells.
【學(xué)位授予單位】：浙江農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S835

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