本文選題：小鼠　切入點：卵母細胞　出處：《山東大學(xué)》2005年博士論文

【摘要】：人胚胎干細胞因在再生醫(yī)學(xué)和人胚胎早期發(fā)育研究等方面潛在的應(yīng)用價值而成為近年來生命科學(xué)的研究熱點,但是人胚胎干細胞研究面臨著嚴重的倫理學(xué)問題和資源匱乏的限制。本課題圍繞卵母細胞激活和胚胎干細胞分離、培養(yǎng)這兩大主題,共進行四部分實驗研究,期望在避免或減少倫理學(xué)問題的情況下,為人胚胎干細胞研究提供較為充足的胚胎來源,為成功建立人胚胎干細胞系奠定實驗基礎(chǔ)�，F(xiàn)分部敘述如下: 第一部分 小鼠卵母細胞孤雌激活的實驗研究 目的:探討化學(xué)激活劑對小鼠卵母細胞的孤雌激活作用以及分離培養(yǎng)孤雌生殖干細胞的可行性。方法:采用有重復(fù)的三因素三水平的正交設(shè)計,觀察乙醇對小鼠卵母細胞的激活作用。乙醇聯(lián)合6-DMAP/嘌呤霉素對小鼠卵母細胞實施激活;采用鈣離子載體A23187(calcium ionophore A23187,A23187)聯(lián)合6-DMAP/嘌呤霉素對小鼠進行激活處理。觀察各種化學(xué)激活劑對小鼠卵母細胞激活效果和孤雌胚胎的發(fā)育潛能。將孤雌囊胚接種于飼養(yǎng)層細胞上,觀察孤雌胚胎在飼養(yǎng)層細胞上的生長情況。結(jié)果:適于小鼠卵母細胞激活和孤雌胚發(fā)育的卵齡是9小時;乙醇濃度為7%;處理時間是5分鐘。但是隨著卵齡增大及乙醇濃度提高,卵母細胞的碎裂率明顯增加。乙醇聯(lián)合6-DMAP/嘌呤霉素后,孤雌胚胎發(fā)育潛能顯著提高。A23187能激活小鼠卵母細胞;與6-DMAP/嘌呤霉素聯(lián)合應(yīng)用能有效地提高孤雌胚胎的發(fā)育潛能。A23187聯(lián)合6-DMAP激活的卵母細胞能發(fā)育到囊胚階段,接種于飼養(yǎng)層后,孤雌囊胚能夠貼壁生長,分離出干細胞克隆,但是孤雌生殖干細胞生長能力明顯較正常胚胎的低下。結(jié)論:乙醇、鈣離子載體A23187或聯(lián)合6-DMAP/嘌呤霉素均能有效地激活小鼠卵母細胞,聯(lián)合應(yīng)用能提高孤雌
[Abstract]:Human embryonic stem cells have become a research hotspot in life sciences in recent years because of their potential application value in regenerative medicine and early development of human embryos.However, human embryonic stem cell research is facing serious ethical problems and resource constraints.This paper focuses on oocyte activation and embryonic stem cell separation and culture, and carries out four parts of experimental research in order to avoid or reduce ethical problems.The research of human embryonic stem cells (ESCs) provides sufficient embryonic sources and lays the experimental foundation for the successful establishment of human embryonic stem cell lines.The current segment is as follows:Experimental study on parthenogenetic activation of mouse oocytesAim: to investigate the effect of chemical activator on parthenogenetic activation of mouse oocytes and the feasibility of isolation and culture of parthenogenetic stem cells.Methods: the effects of ethanol on mouse oocyte activation were observed by orthogonal design with repeated three factors and three levels.Mouse oocytes were activated by ethanol combined with 6-DMAP/ purine mycin, and activated by A23187(calcium ionophore A23187 A23187 and 6-DMAP/ purine mycin.The effects of various chemical activators on oocyte activation and the developmental potential of parthenogenetic embryos were observed.Parthenogenetic blastocysts were inoculated into feeder layer cells to observe the growth of parthenogenetic embryos in feeder layer cells.Results: the oocyte age of mouse oocyte activation and parthenogenetic embryo development was 9 hours, ethanol concentration was 7 and treatment time was 5 minutes.However, with the increase of egg age and ethanol concentration, the cleavage rate of oocytes increased significantly.After ethanol and 6-DMAP/ purine mycin, the developmental potential of parthenogenetic embryos increased significantly. A23187 activated mouse oocytes.Combined with 6-DMAP/ purine mycin can effectively improve the developmental potential of parthenogenetic embryos. A23187 and 6-DMAP activated oocytes can develop to blastocyst stage. After inoculation in feeder layer, parthenogenetic blastocysts can adhere to the wall and isolate stem cell clones.But the growth ability of parthenogenetic stem cells was significantly lower than that of normal embryos.Conclusion: ethanol, calcium carrier A23187 or 6-DMAP/ purine mycin can effectively activate mouse oocytes, and combined use can improve parthenogenetic activity.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R321

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