神经干细胞低温保存的研究

发布时间：2018-03-04 15:31

  本文选题：神经干细胞　切入点：神经球　出处：《大连理工大学》2006年硕士论文　论文类型：学位论文

【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。
[Abstract]:Neural stem cells (NSCs) have become the best choice for the treatment of neurodegenerative diseases and nervous system injuries due to their lack of carcinogenicity after transplantation. Cryopreservation of neural stem cells has become an important subject. At present, cryopreservation of neural stem cells is mainly by slow and quick freezing, and vitrification is a new type of cryopreservation. The simple method of cryopreservation can effectively overcome the shortcoming of slow freezing. On the one hand, the diameter of neural stem cell ball and the types of cryopreservation protectants during the slow freezing of neural stem cells are studied in this paper. On the other hand, the kinds of glass solution and the process of introducing glass solution were studied. In this paper, we first studied the cryopreservation of neural stem cells. By measuring the growth curve of neural stem cells, we determined the best time and state of cryopreservation of neural stem cells. Seven cell spheres of different sizes (30 ~ 50 渭 m in diameter and 80 ~ 100 渭 m in diameter) were used to compare the cryopreservation of seven different concentrations (30 渭 m, 50 渭 m and 80 渭 m DMSO). The best cryopreservation effect was obtained when the diameter of neural stem cells was about 80 渭 m, 100 渭 m DMSO was 8, and the diameter of neural stem cells was about 80 渭 m and 100 渭 m DMSO was 8. The cell viability after resuscitation is about 83%. The different diameter of neural stem cell balls require different concentrations of cryoprotectants. And the close connection between neural stem cells and the interaction between shrinkage signal and diameter size, It affects the cryopreservation and resuscitation of neural stem cells. Secondly, the experimental comparison of neural stem cell vitrification protectants was carried out. The dynamic and intuitive observation on the cold stage and the comparison of neural stem cell resuscitation rate were carried out. The chilled protectant 20v / v) dimethyl sulfoxide (DMSOO) 20g / v) ethylene glycol (EG10) and acetamide (Acetamide) 0.3M sucrose (0.3M) were selected for cryopreservation of neural stem cells (NSCs). Then the operating temperature of the prepared glass protectant during the introduction process was studied. The effect of the time of introduction on the viability of neural stem cells was studied. The experimental temperature of 20 鈩，

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