双酚A对人胚胎干细胞拟胚体发育的影响：初步的基因组学研究

发布时间：2018-06-15 06:48

  本文选题：双酚A + 人胚胎干细胞拟胚体　； 参考：《南京医科大学》2012年硕士论文

【摘要】：研究背景：大量研究利用胚胎干细胞（Embronic stem cell,ES）的体外特性，从细胞毒性、分化抑制和基因表达等不同水平研究胚胎发育及其调节，显示其良好的应用前景。ES悬浮培养后可自发形成拟胚体（Embryoid bodies,EB），分化为包括内胚层、中胚层和外胚层在内的多种类型细胞。EB基本再现了胚胎从原肠运动前到早期原肠运动这段时间的胚胎早期发育过程，并且便于进行各种操作处理，因此EB是研究胚胎早期发育及其调节、遗传和表观遗传、胚层间相互诱导、器官空腔化形成、环境因素对胚胎早期发育的影响等课题的最佳实验模型。 双酚A(Bisphenol A，BPA)作为一种重要的工业原料，广泛存在于塑料制品中，在哺乳动物体内有弱雌激素的活性BPA被广泛用于食品包装材料对人类的健康风险或潜在风险如何，是公众与政府密切关注的问题，也是近20年来持续的研究热点。目前BPA的参考安全摄入剂量为每天50μg/kg，但近年来的动物实验表明即使低于安全摄入剂量，BPA对机体的影响仍然存在。BPA对人类胚胎早期发育的影响、遗传和表观遗传、及其与胎源性疾病的关系，正越来越受关注。 本课题在我中心建立人类胚胎干细胞拟胚体培养系统，以EB为模型、应用基因组学技术，初步研究BPA对胚胎早期发育的影响。本文采用EB系统开展的研究，避免了伦理冲突带来的不便，通过对BPA影响人胚胎早期发育的相关基因表达谱的初步研究，有助于增进对环境内分泌干扰物的研究水平，从遗传和表观遗传学水平深入探讨BPA生殖遗传毒性的分子机制。 研究目的：1、在本中心建立人胚胎干细胞拟胚体培养平台，用于研究人类早期胚胎发育及其调节。2、以人EB为模型，应用基因组学技术，初步研究BPA对胚胎早期发育的影响，从遗传和表观遗传学水平探讨BPA生殖遗传毒性的分子机制。3、作为本课题组研究BPA影响胚胎早期发育和生殖遗传学的一项探索性研究，为今后扩大样本量的实验、增加验证基因的实验、目标基因的功能研究等工作奠定了基础。 研究方法: 一、建立人胚胎干细胞拟胚体培养平台 利用本实验室建立的多株人ES系，建立人EB模型。人ES克隆行AKP染色和TRA-1-81活细胞染色鉴定；通过RT-PCR方法和间接免疫荧光法检测人ES细胞标志性基因表达；检测人ES细胞染色体核型；ES细胞接种至SCID小鼠皮下，观察畸胎瘤形成情况。鉴定ES细胞系后，撤除胎鼠成纤维细胞铺层和碱性成纤维细胞生长因子悬浮培养ES克隆约7天，期间隔天换液，连续观察EB形成情况。培养7天后形成稳定的EB，EB鉴定是通过RT-PCR方法检测三胚层标志基因的表达。 二、双酚A影响人EB早期发育的基因组学研究 ES细胞在无胎鼠成纤维细胞铺层和碱性成纤维细胞生长因子的悬浮条件下培养形成EB。实验组：EB形成过程中用10-6mol/L BPA处理；对照组：采用等量DMSO处理。形成EB（ES悬浮培养7天）后送检基因芯片，采用RocheNimbleGen真核生物表达谱芯片检测全基因组的表达谱，对差异基因利用网站(http://bioinfo.capitalbio.com/mas3/)分析其功能、通路，分类GO注解。选择2个差异基因（PTN和FABP7）采用real-time PCR方法验证芯片结果。采用同法处理另一批EB，进一步验证由生物信息学所得的差异基因。为扩大样本量实验、增加验证基因、目标基因的功能研究等工作，奠定了基础。 结果： 一、人胚胎干细胞拟胚体培养平台的建立 1、实验室前期建立ES细胞呈克隆样形态生长，ES细胞碱性磷酸酶染色和活细胞多能因子TRA-1-81染色呈阳性并表达ES细胞特有的标志性基因，ES细胞具有正常二倍体核型和在SCID鼠体内形成畸胎瘤的能力，表明本实验室前期建立的ES系符合国际检测标准，适合用来形成EB； 2、生长良好、大小基本一致的ES克隆块于未加bFGF因子的ES培养液中悬浮，定期换液观察，可见批量EB形成； 3、获得EB边缘规整，形态呈圆球状，中间隐约可见囊腔。表达人三个胚层标志基因（AFP/BMP4/Nestin）。 二、双酚A作用于人胚胎干细胞拟胚体系统的基因组表达谱 双酚A实验组与DMSO对照组相比，在经10-6mol/L BPA处理7天后的拟胚体中表达上调2倍及以上的基因共有128个，表达下调2倍及以上的基因共有431个。利用生物分子功能注释系统（Molecule Annotation System，MAS）对这些差异表达基因进行功能分析，发现它们与细胞分化、分子功能、胚层发育及信号传导通路相关。经扩大样本后验证发现双酚A可下调与神经系统发育相关的基因PTN和FABP7。 结论： 1、在本中心成功建立人胚胎干细胞拟胚体培养平台、EB鉴定的技术平台。 2、ES悬浮培养后可自发形成拟胚体，分化为包括内胚层、中胚层和外胚层在内的多种类型细胞，是研究人类早期胚胎发育及其调节的理想工具。 3、本文探索性地研究了BPA影响人胚胎早期发育的表达基因组学改变。双酚A作用于拟胚体，有128个基因表达上调，431个基因表达下调，涉及细胞分化、分子功能、胚层发育及信号传导通路等细胞功能和细胞事件，如PTN和FABP7与神经系统发育相关。 4、本文仅为系列研究的一部分，，但初步的实验为课题组今后扩大样本量的实验、增加验证基因的实验、设计更合适的BPA处理浓度和处理时间、目标基因的功能研究、BPA对胚胎早期发育表观遗传学影响等研究工作奠定了基础。
[Abstract]:BACKGROUND : In vitro , embryonic stem cells ( ES ) are used to study the development and regulation of embryonic stem cells ( ES ) from different levels of cell toxicity , differentiation inhibition and gene expression .

BPA ( BPA ) , as an important industrial raw material , is widely used in plastic products . It is widely used in food packaging materials for human health risks or potential risks . It is a hot topic for the public to pay close attention to the government . However , in recent years animal experiments have shown that the influence of BPA on the organism is still present . The effects of BPA on the early development of human embryos , inheritance and epigenetics , and their relationship with fetal origin diseases are becoming more and more attention .

In this paper , we study the effect of BPA on the early embryo development of human embryonic stem cells by using EB as the model and using genomics . The study of EB system avoids the inconvenience caused by the ethical conflict . Through the preliminary study on the expression profiles of genes related to the early development of BPA , it is helpful to study the molecular mechanism of BPA ' s genetic toxicity from genetic and epigenetics levels .

Objective : 1 . To study the effect of BPA on early embryonic development of human embryonic stem cells by using human EB as a model and to study the molecular mechanism of BPA ' s genetic toxicity .

Study method :

1 . Establishment of human embryonic stem cell pseudo - embryonic culture platform

A human EB model was established by using human ES lines established in this lab . Human ES clones were identified by the staining and TRA - 1 - 81 viable cell staining .
The expression of marker genes of human ES cells was detected by RT - PCR and indirect immunofluorescence assay .
detecting human ES cell chromosome karyotype ;
ES cells were seeded subcutaneously in SCID mice to observe the formation of teratomas . After identification of ES cell lines , the cells were removed from fetal rat fibroblasts and the basic fibroblast growth factor suspension cultured ES clones were cultured for 7 days . After 7 days of culture , stable EB was formed , and EB was identified by RT - PCR .

The Genomics Study on the Early Development of EB in Human EB Affected by Diol A

In the experimental group , the EB formation process was treated with 10 - 6 mol / L BPA during EB formation .
In the control group , an equal amount of DMSO was used to test the gene chip after EB ( ES suspension culture for 7 days ) . The expression profile of the whole genome was analyzed by using the RocheNileGen eukaryotic expression profiling chip . The difference gene was verified by the real - time PCR method .

Results :

1 . Establishment of Human Embryonic Stem Cell Quasi - embryonic Culture Platform

1 . In the early stage of the lab , ES cells were grown in the form of clone - like morphology , ES cells alkaline phosphatase staining and multi - energy factors TRA - 1 - 81 were positive and expressed ES cell - specific marker genes , ES cells had normal diploid karyotype and the ability to form teratomas in SCID mice .


2 . ES clones with good growth and basically consistent size were suspended in ES culture medium without bFGF factors .


3 . The EB edge regularity was obtained , the shape was spherical , and the cystic cavity was invisible in the middle . The three embryonic marker genes ( AFP / BMP4 / Nestin ) were expressed .

Genomic expression profiles of bisphenol A in human embryonic stem cell pseudo - embryonic stem cells

There were a total of 128 genes up - regulated and down - regulated by 2 - fold or more in a 10 - 6 mol / L BPA - treated group compared with the DMSO control group .

Conclusion :

1 . The establishment of human embryonic stem cell pseudo - germ culture platform and EB identification technology platform were successfully established in the center .

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本文编号：2021097