猪成体神经干细胞分离、培养的研究

发布时间：2018-04-04 09:03

本文选题：神经干细胞　切入点：猪　出处：《东北农业大学》2011年硕士论文

【摘要】：神经干细胞用于神经学临床修复和基础理论研究的前提是首先完成神经干细胞的体外分离、培养、纯化并大量扩增。鼠、人中都已成功分离出神经干细胞并已尝试用于动物神经系统损伤等疾病的治疗,尽管在鼠和人上的研究很多,相对于鼠神经干细胞在神经学临床应用上的局限和人神经干细胞在材料来源上的不便,猪作为神经干细胞临床应用和基础研究的模式动物有很大的潜力。成体神经干细胞在细胞替代疗法的研究和应用中具有广阔的前景。成体神经干细胞移植不仅可以避免应用胚胎神经干细胞进行细胞替代治疗所带来的伦理问题,而且还可解决由异体或异种移植而导致的免疫排斥问题,使临床应用成为可能。目前,猪成体神经干细胞系尚未被建立起来。本研究旨在探索如何从成猪脑组织中分离培养神经干细胞,观察神经干细胞生长特性和体外增殖的特点。主要的实验方法和实验结果如下: 第一部分:选取适用于人或/和鼠的4种培养体系筛选适用于猪的成体神经干细胞的培养条件。结果表明无血清的悬浮或贴壁培养条件不适用于原代分离猪成体神经干细胞,只有完全培养液中含有血清的DMEM/F12+10% FBS+20ng/ml bFGF+20ng/ml hEGF+103U/ml LIF培养体系支持猪成体神经干细胞的存活和增殖。第二部分:选取3月龄成猪脑室下区(SVZ)、海马齿状回颗粒细胞下区(SGZ)、嘴侧迁移通路(RMS)、海马角(HH)4个脑区分离培养成体神经干细胞,检测可以分离出猪成体神经干细胞的部位。结果表明4个区域均含有成体神经干细胞,可进行原代分离,但无法继代培养。第三部分:探讨猪龄对神经干细胞培养的影响。结果显示3月龄猪SVZ来源的成体神经干细胞在P3呈现的是完全分化的贴壁形态,而1日龄新生猪SVZ来源的成体神经干细胞在P3呈现的是悬浮生长的神经球形态,说明1日龄新生猪SVZ比3月龄成猪SVZ获得P3神经球的效率高。
[Abstract]:The premise of neural stem cells for clinical nerve repair and basic theoretical research is to isolate, culture, purify and amplify neural stem cells in vitro.Neural stem cells have been successfully isolated from both rats and humans and have been tried to treat diseases such as neurological damage in animals, despite much research on mice and humans.Compared with the limitation of clinical application of neural stem cells in rats and the inconvenience of human neural stem cells in material source, pigs have great potential as model animals for clinical application and basic research of neural stem cells.Adult neural stem cells have broad prospects in the research and application of cell replacement therapy.Adult neural stem cell transplantation can not only avoid the ethical problems caused by the application of embryonic neural stem cells in cell replacement therapy, but also solve the problem of immune rejection caused by allogeneic or xenotransplantation, which makes clinical application possible.At present, porcine adult neural stem cell lines have not been established.The purpose of this study was to investigate how to isolate and culture neural stem cells from adult porcine brain tissue and observe the characteristics of neural stem cell growth and proliferation in vitro.The main experimental methods and results are as follows:Part one: four culture systems suitable for human and / or mouse were selected to screen the culture conditions of adult neural stem cells suitable for pigs.The results showed that serum-free suspension or adherent culture was not suitable for primary isolation of porcine adult neural stem cells. Only DMEM/F12 10% FBS 20ng/ml bFGF 20ng/ml 20ng/ml 103U/ml LIF containing serum could support the survival and proliferation of porcine adult neural stem cells.The second part: the adult neural stem cells were isolated and cultured in the three month old adult pig subventricular area (SVZ), the subgranular cells of the dentate gyrus (SGZ), the lateral migration pathway (RMSA) and the hippocampal horn (HH), and the location of the porcine adult neural stem cells (NSCs) could be detected.The results showed that all the four regions contained adult neural stem cells, which could be isolated in primary culture, but could not be subcultured.Part three: to investigate the effect of pig age on neural stem cell culture.The results showed that 1 day old newborn pig SVZ was more efficient than 3 month old adult pig SVZ in obtaining P3 neurospheres.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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