粤黄鸡PGCs无饲养层培养体系的建立

发布时间：2018-01-24 03:48

本文关键词： 原始生殖细胞(PGCs) 无饲养层 PKH 胚胎注射　出处：《农业生物技术学报》2017年08期 　论文类型：期刊论文

【摘要】：原始生殖细胞(primordial germ cells,PGCs)是生殖母细胞的前体细胞,作为一种多能干细胞,具有与胚胎干细胞相似的形态及分化潜能,能在体外进行培养、传代,并在一定条件下保持未分化状态及多能性。现阶段PGCs的体外培养体系中往往存在饲养层细胞和血清,为后期研究造成了困难和混淆。为建立粤黄鸡(Gallus gallus)PGCs的体外无饲养层培养体系。本研究通过改进培养体系成分分离纯化PGCs并进行体外培养。结果显示,所得无饲养层培养的PGCs直径为6~8μm,生长曲线呈现"S"形,群体倍增时间(3.75±0.15)d,结果表明,PGCs具有良好的增殖生长状态;对分离纯化的PGCs特异性基因胚胎阶段特异性表面抗原(stage specific embryonic antigen-1,SSEA-1)和类无精症缺失基因(deleted in azoospermia-like,DAZL)进行免疫荧光鉴定,结果显示二者在PGCs均有表达,经化学免疫染色的细胞可观察到绿色的荧光;将无饲养层培养的PGCs用PKH26标记,注射到孵化至17~20期受体鸡胚的背主动脉中,继续孵化至第5.5天,结果显示,分离纯化并通过PKH26标记的鸡胚血液PGCs仍然具有PGCs的特性,可随鸡胚的血液循环定殖于鸡胚性腺中。本研究优化了PGCs体外培养的条件,建立无饲养层无血清培养体系,为家禽PGCs应用于现代保种技术、转基因技术以及家禽的发育生物学等研究领域提供了重要的技术支持。
[Abstract]:Primordial germ cells (PGCs) are progenitor cells of reproductive mother cells, which are multipotent stem cells. It has similar morphological and differentiation potential to embryonic stem cells and can be cultured and subcultured in vitro. Under certain conditions undifferentiated state and pluripotency were maintained. Feeder layer cells and serum were often found in the present culture system of PGCs in vitro. In order to establish Gallus gallus of Yuehuang Chicken, it is difficult and confusing for the later study. PGCs culture system without feeder layer in vitro. In this study, PGCs was isolated and purified by improved culture system and cultured in vitro. The results showed that. The diameter of PGCs was 6 渭 m, the growth curve was "S" shape, and the population doubling time was 3.75 卤0.15 days. The results showed that the growth curve was of "S" shape and the population doubling time was 3.75 卤0.15 days. PGCs had good proliferation and growth state. The stage specific embryonic antigen-1 of PGCs specific gene was isolated and purified. SSEA-1) and deleted in azoospermia-like DAZL) were identified by immunofluorescence. The results showed that both of them were expressed in PGCs, and green fluorescence could be observed in chemically stained cells. The cultured PGCs was labeled with PKH26 and incubated into the dorsal aorta of the 170-stage recipient chicken embryo and incubated to 5.5d. The chicken embryo blood PGCs, which was isolated and purified by PKH26, still has the characteristics of PGCs. The blood circulation of chicken embryo can be colonized in the gonad of chicken embryo. This study optimized the condition of PGCs culture in vitro and established a serum-free culture system without feeding layer. Transgenic technology and poultry developmental biology provide important technical support.
【作者单位】：佛山科学技术学院生命科学与工程学院;
【基金】：国家自然科学基金(No.31201871)
【分类号】：S831
【正文快照】： 原始生殖细胞(primordial germ cells,PGCs)是各级生殖细胞的祖细胞,同时也是能够发育成精子和卵母细胞的干细胞。PGCs首先在鸡(Gallusgallus)胚的生殖腺中发现,在体外适宜培养条件下,其形态、表面标志和分化潜能均类似于胚胎干细胞(embryonic stem cells,ES)细胞(Matsui et a

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