鸡胚胎原始生殖细胞建系能力的研究
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摘要
胚胎原始生殖细胞(Embryonic Primordial Germ Cells,EPGCs)是生殖母细胞的前体细胞,是精子或卵子的祖先细胞,在多能干细胞研究领域中已成为一个新的干细胞资源,亦是近年来干细胞研究的又一个热点。基于目前国内外对鸡胚胎原始生殖细胞(EPGCs)正处于起步的研究现状,本研究对这一胚胎生殖系干细胞进行了系统的探索性研究。在鸡胚发育的第14期血液中、第19期和第28期的生殖腺中采用不同的分离提取方法分别获取EPGCs,并进行体外培养,以探讨分离培养EPGCs的适宜时期和方法;系统地探索了EPGCs的体外培养体系、传代方法和条件、不同冷冻体系对鸡EPGCs冷冻保存的效果以及外源性细胞因子mLIF、hSCF、bFGF和hIL-11对体外培养的鸡EPGCs增殖和分化的影响,以期筛选出鸡EPGCs合适的体外培养体系和冷冻保存条件;同时利用不同特异性化学物质定向诱导EPGCs向脂肪细胞、神经元样细胞分化,以及尝试对EPGCs单细胞进行体外培养克隆传代、检测其形态特征、表面标记及体外分化等特征特性,为建立EPGCs干细胞系以及体外研究胚胎发育、基因组学研究、药物筛选等提供有价值的参考依据。
研究结果主要归纳为以下几个方面:
1.采用Ficoll密度梯度离心法+酶解法、单独EDTA-酶解法两种方法,分别提取第14期血液(孵化53小时)、第19期(孵化72小时)和第28期(孵化132小时)生殖腺中的鸡EPGCs,比较在相同的体外培养条件下两种分离方法获取三个发育时期的鸡EPGCs数量、存活率和存活时间的差异。结果表明:单独EDTA-酶解法
Embryonic primordial germ cells (EPGCs) are the embryonic precursors of the germ cells (ovum and spermatozoa) and adult gametes, which retain developmental pluripotency as one of new embryonic stem cell resources, are crucial for related stem cells research. The aim of this study was to evaluate the efficiency of isolation and culture of EPGCs from various tissues of chicken embryos at specific developmental stages, explore proper conditions and factors for the cultivation and passage of EPGCs, compare the effect of exogenous cell factors such as mLIF, hSCF, bFGF and hIL-11 on EPGCs proliferation and differentiation and discuss the control mechanism of those cell factors , explore the capability of EPGCs to differentiate into adipocytes and neuron-like cells under different experimental conditions in vitro. Finally, attempt to establish in terms of chicken single EPGC line and determine the pluripotency of EPGCs in detail as configuration, markers of pluripotent status and characteration of differentiation in vitro. Thus, the obtained experimental evidence will contribute to establishment chicken EPGCs-derived ES lines, provide valuble reference for genomic study and medicine invention.
The main results were as follows:
1. EPGCs were derived in chicken embryos at specific developmental stages including: the circulating blood at stage 14 embryos (hatched for 48~52hrs), the genital ridge at
研究结果主要归纳为以下几个方面:
1.采用Ficoll密度梯度离心法+酶解法、单独EDTA-酶解法两种方法,分别提取第14期血液(孵化53小时)、第19期(孵化72小时)和第28期(孵化132小时)生殖腺中的鸡EPGCs,比较在相同的体外培养条件下两种分离方法获取三个发育时期的鸡EPGCs数量、存活率和存活时间的差异。结果表明:单独EDTA-酶解法
Embryonic primordial germ cells (EPGCs) are the embryonic precursors of the germ cells (ovum and spermatozoa) and adult gametes, which retain developmental pluripotency as one of new embryonic stem cell resources, are crucial for related stem cells research. The aim of this study was to evaluate the efficiency of isolation and culture of EPGCs from various tissues of chicken embryos at specific developmental stages, explore proper conditions and factors for the cultivation and passage of EPGCs, compare the effect of exogenous cell factors such as mLIF, hSCF, bFGF and hIL-11 on EPGCs proliferation and differentiation and discuss the control mechanism of those cell factors , explore the capability of EPGCs to differentiate into adipocytes and neuron-like cells under different experimental conditions in vitro. Finally, attempt to establish in terms of chicken single EPGC line and determine the pluripotency of EPGCs in detail as configuration, markers of pluripotent status and characteration of differentiation in vitro. Thus, the obtained experimental evidence will contribute to establishment chicken EPGCs-derived ES lines, provide valuble reference for genomic study and medicine invention.
The main results were as follows:
1. EPGCs were derived in chicken embryos at specific developmental stages including: the circulating blood at stage 14 embryos (hatched for 48~52hrs), the genital ridge at
引文
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