卵母细胞激活技术与胚胎干细胞分离、培养

发布时间：2018-04-02 15:32

本文选题：小鼠　切入点：卵母细胞　出处：《山东大学》2005年博士论文

【摘要】：人胚胎干细胞因在再生医学和人胚胎早期发育研究等方面潜在的应用价值而成为近年来生命科学的研究热点,但是人胚胎干细胞研究面临着严重的伦理学问题和资源匮乏的限制。本课题围绕卵母细胞激活和胚胎干细胞分离、培养这两大主题,共进行四部分实验研究,期望在避免或减少伦理学问题的情况下,为人胚胎干细胞研究提供较为充足的胚胎来源,为成功建立人胚胎干细胞系奠定实验基础。现分部叙述如下: 第一部分小鼠卵母细胞孤雌激活的实验研究目的:探讨化学激活剂对小鼠卵母细胞的孤雌激活作用以及分离培养孤雌生殖干细胞的可行性。方法:采用有重复的三因素三水平的正交设计,观察乙醇对小鼠卵母细胞的激活作用。乙醇联合6-DMAP/嘌呤霉素对小鼠卵母细胞实施激活;采用钙离子载体A23187(calcium ionophore A23187,A23187)联合6-DMAP/嘌呤霉素对小鼠进行激活处理。观察各种化学激活剂对小鼠卵母细胞激活效果和孤雌胚胎的发育潜能。将孤雌囊胚接种于饲养层细胞上,观察孤雌胚胎在饲养层细胞上的生长情况。结果:适于小鼠卵母细胞激活和孤雌胚发育的卵龄是9小时;乙醇浓度为7%;处理时间是5分钟。但是随着卵龄增大及乙醇浓度提高,卵母细胞的碎裂率明显增加。乙醇联合6-DMAP/嘌呤霉素后,孤雌胚胎发育潜能显著提高。A23187能激活小鼠卵母细胞;与6-DMAP/嘌呤霉素联合应用能有效地提高孤雌胚胎的发育潜能。A23187联合6-DMAP激活的卵母细胞能发育到囊胚阶段,接种于饲养层后,孤雌囊胚能够贴壁生长,分离出干细胞克隆,但是孤雌生殖干细胞生长能力明显较正常胚胎的低下。结论:乙醇、钙离子载体A23187或联合6-DMAP/嘌呤霉素均能有效地激活小鼠卵母细胞,联合应用能提高孤雌
[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.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R321

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