小鼠胚胎干细胞分离培养及其生物学特性的研究

【作者】 谢安；

【导师】 汪泱；

【作者基本信息】 南昌大学 ， 免疫学， 2006， 硕士

【摘要】 目的： 胚胎干细胞(embryonic stem cells，ESCs)是由动物或人植入前胚胎的内细胞团(inner cell mass，ICM)或原始生殖细胞(primordial germ cells，PGCs)分离出来，经体外分化抑制培养筛选出的具有全能性的胚胎细胞。ESCs是研究哺乳动物早期胚胎发生，如细胞组织分化、基因表达调控等的一个理想模型，还可为未来再生医学细胞替代疗法和组织器官移植等提供无尽的供体来源。本实验旨在研究如何从BALB／C小鼠的囊胚中分离出具有全能性的胚胎干细胞，并对胚胎干细胞集落进行培养、鉴定，以了解胚胎干细胞生物学特性，为掌握小鼠ESCs分离培养技术及将来进一步研究人ESCs打下基础。 方法： 1、收集BALB／C小鼠3.5d胚龄的囊胚，分离ICM细胞进行培养，以小鼠原代胚胎成纤维细胞(primary mouse embryonic fibroblast，PMEF)作为饲养层，倒置显微镜下观察并记录集落的形成及生长情况，并进行消化传代培养。 2、对细胞集落进行碱性磷酸酶(alkaline pHospHatase，AKP)、特殊期胚胎抗原(stage-specific embryonic antigen，SSEA)-1等ESCs标志物检测，并观察其体外向成熟细胞自然分化及向神经细胞定向诱导分化的潜能。 3、分离提取不同分化阶段ESCs的胞浆及胞核蛋白，通过免疫印迹法(Western Blotting)检测ESCs的增殖分化与核因子-κB(nuclear factor κB，NF-κB)活化的关系。 结果： 1、本实验共获得胚泡71个，内细胞团离散后有30例内细胞团出现类ESCs更多还原

【Abstract】 ObjectiveEmbryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass (ICM) of pre-implantation mammal or human embryos and primordial germ cells (PGCs) of embryos. ESCs were established as a suitable model to study the events which occurred in early development of mammalian embryo, such as cell differentiation and gene expression regulation. It is also an ideal source of tissue engineering and cell substitutive therapy of regenerative medicine. The target of this experiment is to investigate how to isolate totipotent mouse ESCs from BALB/C species mice and to analysis some biological characteristics of ESCs. Methods1、 The blastocysts of 3.5d from BALB/C mice were collected, ICM were isolated and cultured on the primary mouse embryonic fibroblast (PMEF) cell feeder layers. The changes of cell colonies were observed by inverted phase contrast microscope and recorded.2、ES-like colonies were continued disaggregated and cultured. The ES-like colonies were identified by detection of some special markers of ESCs, alkaline phosphatase (AKP) and stage-specific embryonic antigen (SSEA) -1, and by observation the ability of differentiation in Vitro.3、 Cytoplasmic fraction and nuclear fraction of ESCs in different stage were extracted and the relationship of the proliferation and differentiation of ESCs with the activation of nuclear factor kB (NF-κB) were analyzed. Results1、In this study, 71 blastcysts of BALB/C mice were collected, 30 ES-like colonies were obtained and most of them could be cultured to more than three passages, one of them was passaged to 12 passages and cultured stablely .2、All the ES-like colonies showed typical morphological characteristics of ESCs, AKP and SSEA-1 positive expression, and could differentiate to neuron-like更多还原