鹅卵泡闭锁过程中氧化应激诱导颗粒细胞自噬的研究

发布时间：2018-08-27 18:05

【摘要】：浙东白鹅是中国肉鹅中著名的优良品种,具有生长速度快、肉质鲜美等特点,然而其就巢性强,产蛋量低,使品种的发展收到严重阻碍。对就巢期的母鹅的卵巢进行观察,发现其中卵泡均发生不同程度的闭锁,众多研究表明,禽类卵泡中颗粒细胞凋亡是导致其卵泡闭锁的主要原因。哺乳动物中的研究显示,细胞凋亡与细胞自噬密切相关,且作用机制复杂。鉴于此,本试验试图从自噬的角度研究卵泡闭锁的相关机制。试验通过透射电镜、免疫组化等方法,对正常生长卵泡和闭锁卵泡中自噬体进行定位与观察,并采用Western blot、荧光定量PCR等技术,对自噬体标记蛋白LC3的表达量进行检测,判断卵泡闭锁是否与细胞自噬相关。同时,通过多个生化指标的测定,检测氧化应激是否通过诱导卵泡细胞的自噬促进卵泡闭锁。此外,在动物试验结果的基础上,通过培养原代鹅卵泡颗粒细胞,建立氧化应激模型,以进一步在细胞水平进行验证。在鹅卵泡颗粒细胞氧化模型的基础上,分别加入Rapamycin和3-methyladenine(3-MA),以检测卵巢颗粒细胞中mTOR信号通路和Beclin-1信号通路,探究氧化应激诱导鹅卵泡颗粒细胞自噬的作用机制。研究结果表明:与生长卵泡相比,闭锁卵泡颗粒细胞中自噬水平升高(P0.05),氧化应激水平升高(P0.05)。通过H2O2诱导颗粒细胞建立氧化模型,检测发现自噬水平显著升高(P0.05)。对氧化应激模型进行3-MA或Rapamycin共处理,发现Rapamycin共处理组LC3-Ⅱ蛋白表达量显著升高(P0.05),自噬体数量显著增多(P0.05),而3-MA对颗粒细胞自噬水平无影响。研究结论:本研究证明了鹅就巢期卵泡闭锁与颗粒细胞自噬相关,明确了氧化应激引起的内环境变化是诱导颗粒细胞自噬的重要因素。且在氧化条件下,mTOR信号通路是诱导颗粒细胞发生自噬的主要途径。
[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.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S835

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