【作者】 侯颖；

【导师】 马学虎；

【作者基本信息】 大连理工大学 ， 化学工程， 2006， 硕士

【摘要】 神经干细胞因其移植后不具有致癌性等优点,已成为治疗神经系统退行性疾病和神经系统损伤的最优选择。为了更有效地利用神经干细胞,神经干细胞的低温保存技术成为一个重要的课题。目前,低温保存神经干细胞主要采用的是慢速冻存,玻璃化法作为一种新型的,简便的冷冻保存方法可以有效地克服慢速冻存的缺点。本文一方面对神经干细胞慢速冻存过程中神经干细胞球的直径尺寸与冷冻保护剂的种类、浓度进行了实验。另一方面对玻璃化溶液的种类,导入过程等方面的问题进行初步的研究。 首先对神经干细胞的慢速冻存进行了研究。通过神经干细胞生长曲线的测定,确定了低温保存神经干细胞的最佳时间及状态。用处于最佳时间状态的单细胞悬液、不同尺寸的细胞球(直径30～50μm球,直径80～100μm球)分别进行了七个不同浓度3%,5%,7%,8%,10%,15%,20%DMSO的冻存比较。从而确定了神经干细胞处于对数生长期后期,神经球直径约80μm～100μm,DMSO浓度为8%时冻存效果最佳,其复苏后细胞活率约83%。不同直径的神经干细胞球对冷冻保护剂的浓度需求是不同的。且神经干细胞间亲密的联系和皱缩信号与直径尺寸共同作用,影响着神经干细胞的冻存复苏。 其次,对神经干细胞玻璃化保护剂进行了实验比较。通过在冷台上动态直观地观察以及神经干细胞复苏率的比较,优选出了适合神经干细胞玻璃化冻存的冷冻保护剂20%(v/v)二甲基亚砜(DMSO)+20%(v/v)乙二醇(EG)+10%(w/v)乙酰胺(Acetamide)+0.3M蔗糖(Sucrose)。然后研究了所配制的玻璃化保护剂导入过程中操作温度、导入时间对细胞活率的影响。实验温度分别选择了室温(20℃-26℃)和冰水混合物(0℃-4℃)的温度下30s、60s、90s、120s和150s不同时间导入对神经干细胞活率的影响。最后确定了在室温下,60s连续导入神经干细胞的活率最高。更多还原

【Abstract】 Neural stem cells (NSCs) hold tremendous potential in the newly emerging field of regenerative medicine, especially for diseased central nervous system, and to be a useful tool for basic scientific research. However, an essential prerequisite for the future widespread applications of NSC is the development of efficient cryopreservation protocols to facilitate their storage and transportation. At present, slow cooling as a conventional method is the adopted method for long-term storage for NSCs. Vitrification is a promising, novel and simple procedure that requires less time and is likely to become safer and more effective than slow cooling. Vitrification solution(VS) needs a combination of high concentrations of cryoprotectant agents, which have very high toxic and osmotic injuries when the VS is loaded and diluted. This paper focuses on the slow cooling and vitrification protocols, respectively.At first, this paper is attempted to cryopreserve the neural stem cell (NSC) spheres with different diameters and bio-states, aiming to improve the survival rate and explore the underlying mechanism of the cryopreserving process. After the growth curve of the NSC was measured by CCK-8 kit, three types of NSCs in logarithmic growth phase, i.e. the single cell suspension, NSC spheres with the diameters of 30~50μm and 80~100μm, respectively, were cryopreserved at the freezing rate of 1 ℃ /min and seven concentrations of DMSO (3%,5%,7%,8%,10%,1.5%,20%). For the NSC sphere with the diameter of 80~100μm and the DMSO concentration of 8%, the survival rate is 82.9%. The subtle "coordination" of NSC sphere and its diameter together affect the survival rate.Secondly, this paper aiming to select vitrification solution(VS) and loading procedure adapting to NSCs. Through observing on the cryomicroscope system and the survival rate comparision of NSCs, the combination with 20%DMSO+20% EG+10%Acetamide+0.3M Sucrose, is suitableto NSCs. After examing five loading time(30s 60s 90s 120s and 150s) and two loading temperatures(20~26℃ and 0~4℃), It is found that 20~26℃ and 60s is optimal for the good loading procedure of NSCs.更多还原