水牛卵丘细胞主要能量供给途径及葡萄糖对其生长特性影响的初探

发布时间：2018-04-21 04:26

本文选题：水牛卵丘细胞 + 葡萄糖　；参考：《广西大学》2017年硕士论文

【摘要】：葡萄糖是动物体内重要的营养物质,直接或间接参与动物体的新陈代谢过程。研究表明葡萄糖是哺乳动物卵泡发育、卵母细胞成熟和胚胎发育的重要能量来源,并且参与了卵丘扩展、卵母细胞成熟以及早期胚胎发育的分子调控过程。本研究首先探讨水牛卵丘细胞能量供给的主要途径,在此基础上,初步探讨葡萄糖对水牛卵丘细胞生长特性的影响,以期为进一步优化卵丘细胞培养条件,改善卵母细胞体外成熟培养体系奠定基础。首先在体外培养的第二代水牛卵丘细胞的培养液中分别加入氧化磷酸化途径抑制剂、磷酸戊糖途径抑制剂以及糖酵解途径抑制剂,观察细胞的生长状态,并利用ATP生物荧光法检测葡萄糖代谢中相关途径被抑制后细胞的产能变化情况。结果显示:氧化磷酸化途径被抑制后,水牛卵丘细胞生长状态正常,细胞产能变化不显著(P0.05)。磷酸戊糖途径被抑制后,水牛卵丘细胞产能减少,但细胞依然维持正常的生长状态。糖酵解途径被抑制后,水牛卵丘细胞的生长状态会受不同程度的影响,细胞产能与其糖酵解途径受抑制程度呈现出梯度变化。糖酵解途径和磷酸戊糖途径被抑制后,细胞的产能均显著减少(P0.05),但糖酵解途径被抑制后细胞产能减少的程度高于磷酸戊糖途径产能减少的程度(61.46%vs 24.7%)。其次,分别利用不同浓度葡萄糖(0 g/L、1 g/L、4.5 g/L和9 g/L)处理水牛卵丘细胞48 h,探讨葡萄糖对水牛卵丘细胞生长特性的影响,包括细胞生长趋势、细胞倍增时间、细胞增殖水平、细胞凋亡情况和细胞对葡萄糖的利用率等。结果显示:不同浓度葡萄糖作用下水牛卵丘细胞形态无明显区别,细胞生长曲线均呈现“S”型。但细胞培养液中不含葡萄糖,水牛卵丘细胞生长趋势相对缓慢,增殖能力较弱。1 g/L和4.5 g/L浓度葡萄糖处理组细胞生长趋势相似,与对照组相比,两组细胞倍增时间缩短,细胞增殖能力极显著提高(P0.01),以及细胞凋亡率显著下降(P0.05),但4.5 g/L处理组的水牛卵丘细胞对葡萄糖利用率极显著高于1 g/L处理组(P0.01)。细胞培养液中添加9 g/L葡萄糖,在一定时间内有刺激卵丘细胞增殖的效应,但随作用时间延长此效应消失,并出现抑制细胞增殖的现象。利用荧光定量PCR检测水牛卵丘细胞糖酵解相关基因mRNA表达情况,结果发现:4.5g/L处理组细胞糖酵解相关基因HK、PFK和LDH的mRNA表达量均显著高于0 g/L、1 g/L和9 g/L处理组。上述结果表明:(1)糖酵解途径和磷酸戊糖途径是体外培养水牛卵丘细胞的主要能量供给途径,其中糖酵解途径的产能作用更为突出,而水牛卵丘细胞极少利用氧化磷酸化途径产能;(2)适宜葡萄糖浓度(4.5 g/L)可促进水牛卵丘细胞增殖,减少细胞凋亡,且有利于上调糖酵解相关基因HK、PFK和LDH的表达,从而提高细胞糖酵解,促进卵丘细胞对葡萄糖的利用。
[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.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823.83

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