兔胚胎干细胞冷冻保存的研究
文献类型：学位论文
| 作者 | 袁晓华 |
| 学位类别 | 硕士 |
| 答辩日期 | 2007-02 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 季维智 |
| 关键词 | 兔胚胎干细胞 慢速冷冻 防冻液 谷氨酰胺 |
| 其他题名 | The Effects of Different Cryoprotectants on the Cryopreservation of Rabbit Embryonic Stem Cells |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 胚胎干细胞(embryonic stem cells, ES细胞)起源于着床前胚胎内的细胞群，对鼠ES细胞研究已经有20多年，但直到1998年才首次报道从人的胚胎中获得ES细胞，2006年本实验室从兔体外受精胚胎的内细胞团分离建立了兔胚胎干细胞系RF。ES细胞是能在体外长期培养，高度未分化的全能细胞系，可在适合的条件下分化为胎儿或成体的各种类型的组织细胞。根据这一特性，它们可用于再生细胞或组织移植。胚胎干细胞的成功冻存是其应用于临床的前提。成功的冻存是在冷冻、解冻和复苏培养过程中，细胞具有较高的存活率，且仍能保持胚胎干细胞的自我更新和全能性的特性。目前除了小鼠ES细胞用常规慢速冷冻方法可以达到95%以上的未分化集落复苏率外(Yao & Yuan, 2005)，其它物种尤其是灵长类的许多ES细胞系用常规慢速冷冻方法的复苏率极低，极大地限制了这些细胞的临床应用。为提高兔胚胎干细胞RF在慢速冷冻中的保存效果, 本研究比较了二甲基亚砜(DMSO)和乙二醇（ethylene glycol，EG）对兔胚胎干细胞冷冻保护效果。对冷冻复苏后的细胞进行台盼蓝染色，并研究其胚胎干细胞的分子特性，结果表明， DMSO比EG具有更好的冷冻保护效果。再在以10% DMSO为基础的防冻液中添加膜稳定剂海藻糖或谷氨酰胺，细胞冷冻复苏后结果显示, 谷氨酰胺对兔胚胎干细胞有明显的冷冻保护作用，使细胞存活率从71%提高到83.7%。当谷氨酰胺浓度为0、5、10、20、40mmol/L分别加入防冻液中后，20mmol/L的谷氨酰胺具有最佳的冷冻保护效果。以上结果得出兔胚胎干细胞慢速冷冻的防冻液改进配方为：胚胎干细胞培养液中添加10% DMSO+20 mmol/L谷氨酰胺。 |
| 英文摘要 | Embryonic stem (ES) cells are derived from the inner cell mass of blastocysts and have the potential to differentiate into all cell types. So the ES cell lines are valuable for research in developmental biology and transplantation therapy. Non-human ES cell lines can be used as a model system in preclinical experiments to examine the clinical applications of human ES cells. Science the first reported establishment of ES cells derived from mouse embryos in 1981, several ES cell lines have been reported from human, monkey and rat. Recently, rabbit ES cells were derived from in vitro fertilized blastocysts of rabbit in our lab. Satisfactory cryopreservation technologies are crucial for the application of rabbit ES cells. However, the application of slow-cooling cryopreservation protocols to rabbit ES cells was not as efficient as mouse ES cells though both of them were cryopreserved by in single cell suspension. An effective freezing-thawing technique is crucial for the clinical application of newly derived rabbit embryonic stem (ES) cells. The aim of this study was try to find an optimal cryopreservation protocol for rabbit embryonic stem cells using slow freezing-rapid thawing without a programmable freezer. We tested the effects of the following cryoprotective agents (CPAs) on the post-thaw survival, proliferation and differentiation capacity of rabbit embryonic stem cells: ethylene glycol (EG), dimethyl sulphoxide (Me2SO, DMSO), trehalose and glutamine. Trypan blue exclusion tests showed that, among the CPA treatments in this study, EG was more toxic to rabbit embryonic stem cells than DMSO. The highest survival rate (83.7%) was obtained when the rabbit embryonic stem cells were cryopreserved with 10%DMSO and 20 mmol/L glutamine in the ES cell culture media. Thawed ES cells kept their pluripotency and differentiation potential. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6114]  |
| 专题 | 昆明动物研究所_生殖与发育生物学 |
| 推荐引用方式 GB/T 7714 | 袁晓华. 兔胚胎干细胞冷冻保存的研究[D]. 北京. 中国科学院研究生院. 2007. |
入库方式： OAI收割
来源：昆明动物研究所
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