水牛卵泡颗粒细胞自噬及其调控对细胞增殖的影响研究

发布时间：2018-06-19 02:23

本文选题：水牛颗粒细胞 + 卵泡发育　；参考：《广西大学》2015年硕士论文

【摘要】：哺乳动物卵泡发育是一个极为复杂且受到精密调控的生理过程。颗粒细胞作为卵泡的一种主要体细胞,在卵泡发育和卵母细胞成熟过程中起到重要作用,其功能状态决定卵泡的命运。细胞自噬是Ⅱ类程序性细胞死亡,由自噬引起的细胞死亡,可双重调节细胞的死亡和存活,在一定程度上能扭转细胞凋亡的命运,成为细胞存活的促进性因素。目前有关大鼠等动物卵巢颗粒细胞自噬已有许多研究进展,但对水牛颗粒细胞自噬及其与凋亡的关系研究较少。本研究首先按照不同划分标准对卵泡进行分类,包括卵泡直径大小和卵泡闭锁程度,探究卵泡发育阶段与颗粒细胞自噬和凋亡的关系,分析卵泡颗粒细胞自噬、凋亡相关基因的表达规律。然后,利用雷帕霉素作为mTOR途径的抑制剂调控自噬,研究其对体外培养水牛颗粒细胞自噬、凋亡、生长曲线、细胞周期及相关基因表达的影响,初步探索雷帕霉素调控自噬对细胞生长增殖的作用,为进一步优化水牛颗粒细胞体外培养条件奠定基础。1、不同发育阶段水牛卵泡颗粒细胞自噬及凋亡的分析：采用GFP-LC3转染、Annexin V-FITC/PI双染检测不同大小和不同闭锁程度水牛卵泡颗粒细胞的自噬和凋亡状态。结果显示,两种卵泡分类方法试验中,与小卵泡和中卵泡颗粒细胞相比,大卵泡颗粒细胞的自噬率和非活细胞率最高,凋亡率最低；与健康和早期闭锁卵泡颗粒细胞相比,晚期闭锁卵泡颗粒细胞的自噬率和凋亡率较高。运用qRT-PCR方法分析不同大小和不同闭锁程度水牛卵泡颗粒细胞中Atg3、LC3、BECN1等自噬相关基因和Bcl-2、Bax、P53等凋亡相关基因的表达,结果显示,各基因在不同大小卵泡和不同闭锁程度卵泡颗粒细胞中均有表达。与小和中卵泡中的颗粒细胞相比,Atg3、 Atg5、Atg7、 Atg12、BECN1、LC3自噬相关基因在大卵泡颗粒细胞中的表达显著增加(P0.05), ULK1基因表达则最低,差异显著(P0.05)。Atg3、 Atg5、Atg12、BECN1和LC3基因在晚期闭锁卵泡颗粒细胞中的表达最高,随着卵泡闭锁呈现出显著升高(P0.05); Atg7和ULK1基因在健康卵泡颗粒细胞中表达量最高,随着卵泡闭锁程度的增加,二者的表达量逐渐降低。LC3蛋白在成熟卵泡中的表达比原始卵泡中多,与卵泡发育程度呈正相关。2、调控白噬对体外培养水牛颗粒细胞增殖的影响：首先采用透射电镜对体外培养的水牛颗粒细胞进行形态学观察,发现正常培养条件下多种状态的细胞并存,发生白噬的细胞中可观察到自噬体和自噬溶酶体。其次,添加雷帕霉素处理体外培养的水牛颗粒细胞,与添加10%血清DMEM培养的颗粒细胞相比,发现细胞自噬率和凋亡率均显著升高(P0.05)。流式细胞仪分析细胞周期结果显示,雷帕霉素处理后,G0/G1期细胞比例增加,S期和G2/M期细胞比例减少。运用qRT-PCR方法分析雷帕霉素处理后Atg3、Atg5、ULK1等自噬相关基因以及Bcl-2、Bax、P53等凋亡相关基因的表达,发现在0.2nM处理条件下,与添加10%血清DMEM培养的颗粒细胞相比,Atg3、Atg5、ULK1等自噬相关基因表达均有明显升高(P0.05),无剂量依赖性关系。Bax、Caspase3、P53促凋亡基因的表达随雷帕霉素处理浓度的增加而升高,抑凋亡基因Bcl-2表达量随雷帕霉素浓度的减少而降低,Fas基因表达差异不显著(P0.05)。以上研究结果表明,生长过程中的水牛卵泡颗粒细胞发生自噬和凋亡,随着卵泡直径的增大以及闭锁程度的加深,颗粒细胞的自噬率和凋亡率逐渐上升；雷帕霉素通过抑制mTOR途径可诱导体外培养的水牛颗粒细胞发生自噬,细胞自噬与凋亡共同调控颗粒细胞生长。
[Abstract]:Mammalian follicle development is a very complex and precisely regulated physiological process. Granulosa cells act as a major body cell of the follicle and play an important role in follicular development and oocyte maturation. Their functional state determines the fate of the follicles. Cell autophagy is a class of programmed cell death, which is caused by autophagy. Death, which can double regulate cell death and survival, can reverse the fate of cell apoptosis to a certain extent, and become a promoting factor for cell survival. At present, there have been a lot of advances in autophagy in rat ovarian granulosa cells, but there are few studies on the relationship between autophagy and apoptosis of Buffalo granulosa cells. Different criteria were used to classify follicles, including the size of follicle diameter and the degree of follicle atresia, the relationship between the stage of follicle development and the autophagy and apoptosis of granulosa cells, the analysis of autophagy and the expression of apoptosis related genes in the follicle granulosa cells. Then, using rapamycin as a mTOR pathway inhibitor to regulate autophagy and study its effect on the body. To cultivate the autophagy, apoptosis, growth curve, cell cycle and related gene expression of buffalo granulosa cells, the effect of rapamycin on the growth and proliferation of cells was preliminarily explored to further optimize the culture conditions of buffalo granulosa cells in vitro, and to analyze the autophagy and apoptosis of the granulosa cells of buffalo follicles at different developmental stages:.1 The autophagy and apoptosis state of buffalo follicle granulosa cells with different sizes and different atresia levels were detected by GFP-LC3 transfection and Annexin V-FITC/PI double staining. The results showed that, compared with the small follicle and the medium follicle granulosa cells, the rate of autophagy and non living cells was the highest and the rate of apoptosis was the lowest in the two types of follicle classification. Compared with healthy and early atresia follicle granulosa cells, the autophagy rate and apoptosis rate of late atresia follicle granulosa cells were higher. The expression of autophagy related genes, such as Atg3, LC3, BECN1, and Bcl-2, Bax, P53, and other genes in Buffalo follicle granulosa cells of different sizes and atresia were analyzed by qRT-PCR method. The results showed that the genes were related to each gene. The expression of Atg3, Atg5, Atg7, Atg12, BECN1, LC3 related genes in the large follicle granulosa cells increased significantly (P0.05), and the expression of ULK1 gene was the lowest, and the difference was significant (P0.05).Atg3, Atg5, Atg12, Atg12, and Atg12, compared with granular cells in small and middle follicles. The expression of 3 gene in advanced atresia follicle granulosa cells was the highest, with follicle atresia (P0.05), and Atg7 and ULK1 genes expressed in the healthy follicle granulosa cells. With the increase of follicle atresia, the expression of the two was gradually reduced by the expression of.LC3 egg white in the mature follicle than in the original follicle. The effect of the degree of bubble development on the proliferation of buffalo granulosa cells in vitro was regulated by.2. First, transmission electron microscopy was used to observe the morphology of buffalo granulocyte in vitro. It was found that the cells of various states were coexistence under normal culture conditions, and autophagosomes and autophagosomes could be observed in white macrophages. After adding rapamycin to treat the cultured buffalo granulosa cells, the autophagy rate and the apoptosis rate were significantly increased (P0.05) compared with the granulosa cells cultured with 10% serum DMEM. The flow cytometry analysis showed that after rapamycin treatment, the proportion of G0/G1 phase cells increased, and the proportion of S and G2/M phase cells decreased. The expression of autophagy related genes, such as Atg3, Atg5, ULK1, as well as Bcl-2, Bax, P53 and other apoptosis related genes after rapamycin treatment was analyzed by qRT-PCR method. The expression of autophagy related genes, such as Atg3, Atg5, ULK1, and so on, was significantly increased under the condition of 0.2nM treatment. The expression of autophagy related genes, such as Atg3, Atg5, ULK1, and so on, was significantly higher than that of 10% serum DMEM cultured granulosa cells. The expression of.Bax, Caspase3 and P53 increased with the increase of the concentration of rapamycin, and the expression of Bcl-2 was decreased with the decrease of rapamycin concentration, and the difference of Fas gene expression was not significant (P0.05). The above results showed that the buffalo follicle granulosa cells were autophagy and apoptosis in the growth process, with the follicle straight. The autophagy and apoptosis rate of granulosa cells increased gradually, and rapamycin could induce autophagy in the cultured buffalo granulosa cells in vitro by inhibiting the mTOR pathway, and the cell autophagy and apoptosis co regulated the growth of granulosa cells.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823.83

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