發(fā)布時(shí)間：2018-06-19 02:23

  本文選題：水牛顆粒細(xì)胞 + 卵泡發(fā)育　； 參考：《廣西大學(xué)》2015年碩士論文

【摘要】：哺乳動(dòng)物卵泡發(fā)育是一個(gè)極為復(fù)雜且受到精密調(diào)控的生理過程。顆粒細(xì)胞作為卵泡的一種主要體細(xì)胞,在卵泡發(fā)育和卵母細(xì)胞成熟過程中起到重要作用,其功能狀態(tài)決定卵泡的命運(yùn)。細(xì)胞自噬是Ⅱ類程序性細(xì)胞死亡,由自噬引起的細(xì)胞死亡,可雙重調(diào)節(jié)細(xì)胞的死亡和存活,在一定程度上能扭轉(zhuǎn)細(xì)胞凋亡的命運(yùn),成為細(xì)胞存活的促進(jìn)性因素。目前有關(guān)大鼠等動(dòng)物卵巢顆粒細(xì)胞自噬已有許多研究進(jìn)展,但對(duì)水牛顆粒細(xì)胞自噬及其與凋亡的關(guān)系研究較少。本研究首先按照不同劃分標(biāo)準(zhǔn)對(duì)卵泡進(jìn)行分類,包括卵泡直徑大小和卵泡閉鎖程度,探究卵泡發(fā)育階段與顆粒細(xì)胞自噬和凋亡的關(guān)系,分析卵泡顆粒細(xì)胞自噬、凋亡相關(guān)基因的表達(dá)規(guī)律。然后,利用雷帕霉素作為mTOR途徑的抑制劑調(diào)控自噬,研究其對(duì)體外培養(yǎng)水牛顆粒細(xì)胞自噬、凋亡、生長曲線、細(xì)胞周期及相關(guān)基因表達(dá)的影響,初步探索雷帕霉素調(diào)控自噬對(duì)細(xì)胞生長增殖的作用,為進(jìn)一步優(yōu)化水牛顆粒細(xì)胞體外培養(yǎng)條件奠定基礎(chǔ)。1、不同發(fā)育階段水牛卵泡顆粒細(xì)胞自噬及凋亡的分析：采用GFP-LC3轉(zhuǎn)染、Annexin V-FITC/PI雙染檢測不同大小和不同閉鎖程度水牛卵泡顆粒細(xì)胞的自噬和凋亡狀態(tài)。結(jié)果顯示,兩種卵泡分類方法試驗(yàn)中,與小卵泡和中卵泡顆粒細(xì)胞相比,大卵泡顆粒細(xì)胞的自噬率和非活細(xì)胞率最高,凋亡率最低；與健康和早期閉鎖卵泡顆粒細(xì)胞相比,晚期閉鎖卵泡顆粒細(xì)胞的自噬率和凋亡率較高。運(yùn)用qRT-PCR方法分析不同大小和不同閉鎖程度水牛卵泡顆粒細(xì)胞中Atg3、LC3、BECN1等自噬相關(guān)基因和Bcl-2、Bax、P53等凋亡相關(guān)基因的表達(dá),結(jié)果顯示,各基因在不同大小卵泡和不同閉鎖程度卵泡顆粒細(xì)胞中均有表達(dá)。與小和中卵泡中的顆粒細(xì)胞相比,Atg3、 Atg5、Atg7、 Atg12、BECN1、LC3自噬相關(guān)基因在大卵泡顆粒細(xì)胞中的表達(dá)顯著增加(P0.05), ULK1基因表達(dá)則最低,差異顯著(P0.05)。Atg3、 Atg5、Atg12、BECN1和LC3基因在晚期閉鎖卵泡顆粒細(xì)胞中的表達(dá)最高,隨著卵泡閉鎖呈現(xiàn)出顯著升高(P0.05); Atg7和ULK1基因在健康卵泡顆粒細(xì)胞中表達(dá)量最高,隨著卵泡閉鎖程度的增加,二者的表達(dá)量逐漸降低。LC3蛋白在成熟卵泡中的表達(dá)比原始卵泡中多,與卵泡發(fā)育程度呈正相關(guān)。2、調(diào)控白噬對(duì)體外培養(yǎng)水牛顆粒細(xì)胞增殖的影響：首先采用透射電鏡對(duì)體外培養(yǎng)的水牛顆粒細(xì)胞進(jìn)行形態(tài)學(xué)觀察,發(fā)現(xiàn)正常培養(yǎng)條件下多種狀態(tài)的細(xì)胞并存,發(fā)生白噬的細(xì)胞中可觀察到自噬體和自噬溶酶體。其次,添加雷帕霉素處理體外培養(yǎng)的水牛顆粒細(xì)胞,與添加10%血清DMEM培養(yǎng)的顆粒細(xì)胞相比,發(fā)現(xiàn)細(xì)胞自噬率和凋亡率均顯著升高(P0.05)。流式細(xì)胞儀分析細(xì)胞周期結(jié)果顯示,雷帕霉素處理后,G0/G1期細(xì)胞比例增加,S期和G2/M期細(xì)胞比例減少。運(yùn)用qRT-PCR方法分析雷帕霉素處理后Atg3、Atg5、ULK1等自噬相關(guān)基因以及Bcl-2、Bax、P53等凋亡相關(guān)基因的表達(dá),發(fā)現(xiàn)在0.2nM處理?xiàng)l件下,與添加10%血清DMEM培養(yǎng)的顆粒細(xì)胞相比,Atg3、Atg5、ULK1等自噬相關(guān)基因表達(dá)均有明顯升高(P0.05),無劑量依賴性關(guān)系。Bax、Caspase3、P53促凋亡基因的表達(dá)隨雷帕霉素處理濃度的增加而升高,抑凋亡基因Bcl-2表達(dá)量隨雷帕霉素濃度的減少而降低,Fas基因表達(dá)差異不顯著(P0.05)。以上研究結(jié)果表明,生長過程中的水牛卵泡顆粒細(xì)胞發(fā)生自噬和凋亡,隨著卵泡直徑的增大以及閉鎖程度的加深,顆粒細(xì)胞的自噬率和凋亡率逐漸上升；雷帕霉素通過抑制mTOR途徑可誘導(dǎo)體外培養(yǎng)的水牛顆粒細(xì)胞發(fā)生自噬,細(xì)胞自噬與凋亡共同調(diào)控顆粒細(xì)胞生長。
[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.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S823.83

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 高亞可;陸鳳花;馬帆;喬樹葉;陳施蓓;石德順;;水牛卵巢顆粒細(xì)胞的分離培養(yǎng)及凋亡測定[J];中國畜牧獸醫(yī);2012年10期

2 孫雅婧;郭青龍;;細(xì)胞自噬的研究進(jìn)展[J];藥學(xué)與臨床研究;2012年03期

3 馬克學(xué);席興字;;細(xì)胞自噬的作用機(jī)制及功能[J];生物學(xué)教學(xué);2011年05期

4 鄭航;胡偉;鄭新民;李世文;王行環(huán);;雷帕霉素抑制人前列腺癌細(xì)胞增殖及其作用機(jī)制[J];中華實(shí)驗(yàn)外科雜志;2011年01期

5 陳洪菊;屈藝;母得志;;mTOR信號(hào)通路的生物學(xué)功能[J];生命的化學(xué);2010年04期

6 林鵬飛;郝元斌;郭慧利;劉紅林;芮榮;;豬分離卵泡體外培養(yǎng)過程中Fas/FasL對(duì)顆粒細(xì)胞凋亡的作用[J];動(dòng)物學(xué)研究;2010年03期

7 張丹;王海杰;;缺血缺氧狀態(tài)下細(xì)胞自噬的研究進(jìn)展[J];國際病理科學(xué)與臨床雜志;2009年04期

8 鞏勇;倪宏;陶陸陽;;3-甲基腺嘌呤對(duì)反復(fù)驚厥新生大鼠大腦皮層自噬相關(guān)蛋白Beclin1表達(dá)的影響[J];實(shí)用兒科臨床雜志;2009年16期

9 李佳芮;鄧玉杰;張?jiān)S平;付濤;馬源;郭建軍;劉凱于;;雷帕霉素對(duì)二種鱗翅目昆蟲細(xì)胞自噬和凋亡的影響[J];昆蟲知識(shí);2009年03期

10 李欣志;劉建勛;;缺血/再灌注過程中心肌細(xì)胞自噬研究進(jìn)展[J];中國藥理學(xué)通報(bào);2008年06期

，

本文編號(hào)：2037929