發(fā)布時間：2018-04-21 04:26

  本文選題：水牛卵丘細胞 + 葡萄糖　； 參考：《廣西大學(xué)》2017年碩士論文

【摘要】：葡萄糖是動物體內(nèi)重要的營養(yǎng)物質(zhì),直接或間接參與動物體的新陳代謝過程。研究表明葡萄糖是哺乳動物卵泡發(fā)育、卵母細胞成熟和胚胎發(fā)育的重要能量來源,并且參與了卵丘擴展、卵母細胞成熟以及早期胚胎發(fā)育的分子調(diào)控過程。本研究首先探討水牛卵丘細胞能量供給的主要途徑,在此基礎(chǔ)上,初步探討葡萄糖對水牛卵丘細胞生長特性的影響,以期為進一步優(yōu)化卵丘細胞培養(yǎng)條件,改善卵母細胞體外成熟培養(yǎng)體系奠定基礎(chǔ)。首先在體外培養(yǎng)的第二代水牛卵丘細胞的培養(yǎng)液中分別加入氧化磷酸化途徑抑制劑、磷酸戊糖途徑抑制劑以及糖酵解途徑抑制劑,觀察細胞的生長狀態(tài),并利用ATP生物熒光法檢測葡萄糖代謝中相關(guān)途徑被抑制后細胞的產(chǎn)能變化情況。結(jié)果顯示:氧化磷酸化途徑被抑制后,水牛卵丘細胞生長狀態(tài)正常,細胞產(chǎn)能變化不顯著(P0.05)。磷酸戊糖途徑被抑制后,水牛卵丘細胞產(chǎn)能減少,但細胞依然維持正常的生長狀態(tài)。糖酵解途徑被抑制后,水牛卵丘細胞的生長狀態(tài)會受不同程度的影響,細胞產(chǎn)能與其糖酵解途徑受抑制程度呈現(xiàn)出梯度變化。糖酵解途徑和磷酸戊糖途徑被抑制后,細胞的產(chǎn)能均顯著減少(P0.05),但糖酵解途徑被抑制后細胞產(chǎn)能減少的程度高于磷酸戊糖途徑產(chǎn)能減少的程度(61.46%vs 24.7%)。其次,分別利用不同濃度葡萄糖(0 g/L、1 g/L、4.5 g/L和9 g/L)處理水牛卵丘細胞48 h,探討葡萄糖對水牛卵丘細胞生長特性的影響,包括細胞生長趨勢、細胞倍增時間、細胞增殖水平、細胞凋亡情況和細胞對葡萄糖的利用率等。結(jié)果顯示:不同濃度葡萄糖作用下水牛卵丘細胞形態(tài)無明顯區(qū)別,細胞生長曲線均呈現(xiàn)“S”型。但細胞培養(yǎng)液中不含葡萄糖,水牛卵丘細胞生長趨勢相對緩慢,增殖能力較弱。1 g/L和4.5 g/L濃度葡萄糖處理組細胞生長趨勢相似,與對照組相比,兩組細胞倍增時間縮短,細胞增殖能力極顯著提高(P0.01),以及細胞凋亡率顯著下降(P0.05),但4.5 g/L處理組的水牛卵丘細胞對葡萄糖利用率極顯著高于1 g/L處理組(P0.01)。細胞培養(yǎng)液中添加9 g/L葡萄糖,在一定時間內(nèi)有刺激卵丘細胞增殖的效應(yīng),但隨作用時間延長此效應(yīng)消失,并出現(xiàn)抑制細胞增殖的現(xiàn)象。利用熒光定量PCR檢測水牛卵丘細胞糖酵解相關(guān)基因mRNA表達情況,結(jié)果發(fā)現(xiàn):4.5g/L處理組細胞糖酵解相關(guān)基因HK、PFK和LDH的mRNA表達量均顯著高于0 g/L、1 g/L和9 g/L處理組。上述結(jié)果表明:(1)糖酵解途徑和磷酸戊糖途徑是體外培養(yǎng)水牛卵丘細胞的主要能量供給途徑,其中糖酵解途徑的產(chǎn)能作用更為突出,而水牛卵丘細胞極少利用氧化磷酸化途徑產(chǎn)能;(2)適宜葡萄糖濃度(4.5 g/L)可促進水牛卵丘細胞增殖,減少細胞凋亡,且有利于上調(diào)糖酵解相關(guān)基因HK、PFK和LDH的表達,從而提高細胞糖酵解,促進卵丘細胞對葡萄糖的利用。
[Abstract]:Glucose is an important nutrient in animals, directly or indirectly involved in the metabolism of animals. Glucose is an important energy source for follicular development, oocyte maturation and embryonic development in mammals, and is involved in the molecular regulation of cumulus expansion, oocyte maturation and early embryonic development. In this study, the main ways of energy supply of buffalo cumulus cells were discussed. On the basis of this, the effects of glucose on the growth characteristics of buffalo cumulus cells were discussed in order to optimize the culture conditions of cumulus cells. To improve the maturation system of oocytes in vitro. Firstly, oxidative phosphorylation pathway inhibitor, pentose phosphate pathway inhibitor and glycolysis pathway inhibitor were added to the culture medium of the second generation buffalo cumulus cells in vitro to observe the growth state of the cells. ATP biofluorescence assay was used to detect the changes of cell productivity after the inhibition of related pathways in glucose metabolism. The results showed that after the oxidative phosphorylation pathway was inhibited, the growth state of buffalo cumulus cells was normal, and the cell productivity did not change significantly (P 0.05). After pentose phosphate pathway was inhibited, the production capacity of buffalo cumulus cells decreased, but the cells remained normal growth state. After the glycolysis pathway was inhibited, the growth state of buffalo cumulus cells was affected by different degrees, and the cell productivity and the inhibition degree of glycolysis pathway showed a gradient change. After the inhibition of glycolysis pathway and pentose phosphate pathway, the cell productivity decreased significantly, but the cell production capacity decreased after the inhibition of glycolysis pathway was higher than that of pentose phosphate pathway (61.46 vs 24.7%). Secondly, the buffalo cumulus cells were treated with different concentrations of glucose (0 g / L, 1 g / L, 4.5 g / L and 9 g / L) for 48 h, and the effects of glucose on the growth characteristics of buffalo cumulus cells, including cell growth trend, cell doubling time, cell proliferation level, were investigated. Apoptosis and glucose utilization. The results showed that there was no significant difference in the morphology of buffalo cumulus cells under different glucose concentrations, and the cell growth curves were of "S" type. However, the growth trend of buffalo cumulus cells was relatively slow, and the growth trend of buffalo cumulus cells was similar to that of glucose treated with glucose at concentrations of 1.1g / L and 4.5 g / L, compared with the control group, the doubling time of the cells in the two groups was shorter than that in the control group. The cell proliferation ability and apoptosis rate decreased significantly, but the glucose utilization of buffalo cumulus cells in 4.5 g / L treatment group was significantly higher than that in 1 g / L treatment group. When 9 g / L glucose was added to the cell culture medium, the proliferation of cumulus cells was stimulated for a certain period of time, but the effect disappeared with the prolongation of the action time, and the phenomenon of inhibiting cell proliferation appeared. Fluorescence quantitative PCR was used to detect the expression of glycolytic related gene mRNA in buffalo cumulus cells. The results showed that the mRNA expression of glycolytic associated genes HKPK and LDH in the treated group was significantly higher than that in the 0 g / L 1 g / L and 9 g / L groups. These results indicate that the glycolysis pathway and pentose phosphate pathway are the main energy supply pathways for cultured buffalo cumulus cells in vitro. On the other hand, buffalo cumulus cells rarely use oxidative phosphorylation pathway to produce a suitable glucose concentration of 4.5 g / L) which can promote the proliferation of buffalo cumulus cells, reduce apoptosis, and up-regulate the expression of glycolytic genes HKPFK and LDH. Thus, the glycolysis of the cells and the utilization of glucose by cumulus cells were improved.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S823.83

【參考文獻】

相關(guān)期刊論文 前9條

1 張朔;周佳勃;岳順利;張春龍;張利;張宇霆;譚景和;;不同濃度葡萄糖對豬卵母細胞體外成熟及胚胎發(fā)育的影響[J];黑龍江畜牧獸醫(yī);2016年03期

2 楊芳;張寶云;馮光德;向偉;馬云霞;陳航;儲明星;王憑青;;卵巢低氧微環(huán)境調(diào)節(jié)哺乳動物排卵的分子機制[J];遺傳;2016年02期

3 張玲;邵淑敏;陳梅;;糖酵解途徑相關(guān)酶PFK和LDH在人卵巢組織中的表達及意義[J];中國婦幼保健;2014年09期

4 鄒穎;王文梅;朱雅男;楊衛(wèi)東;;CCK-8法評價口腔粘結(jié)材料的體外細胞毒性研究[J];口腔醫(yī)學(xué)研究;2011年08期

5 周佳勃,吳延光,羅明久,韓東,劉麗清,常仲樂,譚景和;影響山羊體外受精的因素[J];動物學(xué)報;2004年02期

6 高敏芝,汪玉寶,吳曉云,顧敦瑜,李忠妹,陸湘,陳淑穎,何志英;小鼠生殖泡期卵母細胞體外培養(yǎng)成熟、受精和胚胎發(fā)育的研究[J];上海實驗動物科學(xué);2003年04期

7 畢春明,陳彥,戴谷,陸金春,李朝軍,張錫然;小鼠卵母細胞生發(fā)泡破裂前的鈣離子波動及分布[J];南京師大學(xué)報(自然科學(xué)版);2000年01期

8 劉U，

本文編號：1780935