本文選題：雙酚A + 人胚胎干細(xì)胞擬胚體��； 參考：《南京醫(yī)科大學(xué)》2012年碩士論文

【摘要】：研究背景：大量研究利用胚胎干細(xì)胞（Embronic stem cell,ES）的體外特性，從細(xì)胞毒性、分化抑制和基因表達(dá)等不同水平研究胚胎發(fā)育及其調(diào)節(jié)，顯示其良好的應(yīng)用前景。ES懸浮培養(yǎng)后可自發(fā)形成擬胚體（Embryoid bodies,EB），分化為包括內(nèi)胚層、中胚層和外胚層在內(nèi)的多種類型細(xì)胞。EB基本再現(xiàn)了胚胎從原腸運(yùn)動(dòng)前到早期原腸運(yùn)動(dòng)這段時(shí)間的胚胎早期發(fā)育過程，并且便于進(jìn)行各種操作處理，因此EB是研究胚胎早期發(fā)育及其調(diào)節(jié)、遺傳和表觀遺傳、胚層間相互誘導(dǎo)、器官空腔化形成、環(huán)境因素對(duì)胚胎早期發(fā)育的影響等課題的最佳實(shí)驗(yàn)?zāi)Ｐ汀?雙酚A(Bisphenol A，BPA)作為一種重要的工業(yè)原料，廣泛存在于塑料制品中，在哺乳動(dòng)物體內(nèi)有弱雌激素的活性BPA被廣泛用于食品包裝材料對(duì)人類的健康風(fēng)險(xiǎn)或潛在風(fēng)險(xiǎn)如何，是公眾與政府密切關(guān)注的問題，也是近20年來持續(xù)的研究熱點(diǎn)。目前BPA的參考安全攝入劑量為每天50μg/kg，但近年來的動(dòng)物實(shí)驗(yàn)表明即使低于安全攝入劑量，BPA對(duì)機(jī)體的影響仍然存在。BPA對(duì)人類胚胎早期發(fā)育的影響、遺傳和表觀遺傳、及其與胎源性疾病的關(guān)系，正越來越受關(guān)注。 本課題在我中心建立人類胚胎干細(xì)胞擬胚體培養(yǎng)系統(tǒng)，以EB為模型、應(yīng)用基因組學(xué)技術(shù)，初步研究BPA對(duì)胚胎早期發(fā)育的影響。本文采用EB系統(tǒng)開展的研究，避免了倫理沖突帶來的不便，通過對(duì)BPA影響人胚胎早期發(fā)育的相關(guān)基因表達(dá)譜的初步研究，有助于增進(jìn)對(duì)環(huán)境內(nèi)分泌干擾物的研究水平，從遺傳和表觀遺傳學(xué)水平深入探討B(tài)PA生殖遺傳毒性的分子機(jī)制。 研究目的：1、在本中心建立人胚胎干細(xì)胞擬胚體培養(yǎng)平臺(tái)，用于研究人類早期胚胎發(fā)育及其調(diào)節(jié)。2、以人EB為模型，應(yīng)用基因組學(xué)技術(shù)，初步研究BPA對(duì)胚胎早期發(fā)育的影響，從遺傳和表觀遺傳學(xué)水平探討B(tài)PA生殖遺傳毒性的分子機(jī)制。3、作為本課題組研究BPA影響胚胎早期發(fā)育和生殖遺傳學(xué)的一項(xiàng)探索性研究，為今后擴(kuò)大樣本量的實(shí)驗(yàn)、增加驗(yàn)證基因的實(shí)驗(yàn)、目標(biāo)基因的功能研究等工作奠定了基礎(chǔ)。 研究方法: 一、建立人胚胎干細(xì)胞擬胚體培養(yǎng)平臺(tái) 利用本實(shí)驗(yàn)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活細(xì)胞染色鑒定；通過RT-PCR方法和間接免疫熒光法檢測人ES細(xì)胞標(biāo)志性基因表達(dá)；檢測人ES細(xì)胞染色體核型；ES細(xì)胞接種至SCID小鼠皮下，觀察畸胎瘤形成情況。鑒定ES細(xì)胞系后，撤除胎鼠成纖維細(xì)胞鋪層和堿性成纖維細(xì)胞生長因子懸浮培養(yǎng)ES克隆約7天，期間隔天換液，連續(xù)觀察EB形成情況。培養(yǎng)7天后形成穩(wěn)定的EB，EB鑒定是通過RT-PCR方法檢測三胚層標(biāo)志基因的表達(dá)。 二、雙酚A影響人EB早期發(fā)育的基因組學(xué)研究 ES細(xì)胞在無胎鼠成纖維細(xì)胞鋪層和堿性成纖維細(xì)胞生長因子的懸浮條件下培養(yǎng)形成EB。實(shí)驗(yàn)組：EB形成過程中用10-6mol/L BPA處理；對(duì)照組：采用等量DMSO處理。形成EB（ES懸浮培養(yǎng)7天）后送檢基因芯片，采用RocheNimbleGen真核生物表達(dá)譜芯片檢測全基因組的表達(dá)譜，對(duì)差異基因利用網(wǎng)站(http://bioinfo.capitalbio.com/mas3/)分析其功能、通路，分類GO注解。選擇2個(gè)差異基因（PTN和FABP7）采用real-time PCR方法驗(yàn)證芯片結(jié)果。采用同法處理另一批EB，進(jìn)一步驗(yàn)證由生物信息學(xué)所得的差異基因。為擴(kuò)大樣本量實(shí)驗(yàn)、增加驗(yàn)證基因、目標(biāo)基因的功能研究等工作，奠定了基礎(chǔ)。 結(jié)果： 一、人胚胎干細(xì)胞擬胚體培養(yǎng)平臺(tái)的建立 1、實(shí)驗(yàn)室前期建立ES細(xì)胞呈克隆樣形態(tài)生長，ES細(xì)胞堿性磷酸酶染色和活細(xì)胞多能因子TRA-1-81染色呈陽性并表達(dá)ES細(xì)胞特有的標(biāo)志性基因，ES細(xì)胞具有正常二倍體核型和在SCID鼠體內(nèi)形成畸胎瘤的能力，表明本實(shí)驗(yàn)室前期建立的ES系符合國際檢測標(biāo)準(zhǔn)，適合用來形成EB； 2、生長良好、大小基本一致的ES克隆塊于未加bFGF因子的ES培養(yǎng)液中懸浮，定期換液觀察，可見批量EB形成； 3、獲得EB邊緣規(guī)整，形態(tài)呈圓球狀，中間隱約可見囊腔。表達(dá)人三個(gè)胚層標(biāo)志基因（AFP/BMP4/Nestin）。 二、雙酚A作用于人胚胎干細(xì)胞擬胚體系統(tǒng)的基因組表達(dá)譜 雙酚A實(shí)驗(yàn)組與DMSO對(duì)照組相比，在經(jīng)10-6mol/L BPA處理7天后的擬胚體中表達(dá)上調(diào)2倍及以上的基因共有128個(gè)，表達(dá)下調(diào)2倍及以上的基因共有431個(gè)。利用生物分子功能注釋系統(tǒng)（Molecule Annotation System，MAS）對(duì)這些差異表達(dá)基因進(jìn)行功能分析，發(fā)現(xiàn)它們與細(xì)胞分化、分子功能、胚層發(fā)育及信號(hào)傳導(dǎo)通路相關(guān)。經(jīng)擴(kuò)大樣本后驗(yàn)證發(fā)現(xiàn)雙酚A可下調(diào)與神經(jīng)系統(tǒng)發(fā)育相關(guān)的基因PTN和FABP7。 結(jié)論： 1、在本中心成功建立人胚胎干細(xì)胞擬胚體培養(yǎng)平臺(tái)、EB鑒定的技術(shù)平臺(tái)。 2、ES懸浮培養(yǎng)后可自發(fā)形成擬胚體，分化為包括內(nèi)胚層、中胚層和外胚層在內(nèi)的多種類型細(xì)胞，是研究人類早期胚胎發(fā)育及其調(diào)節(jié)的理想工具。 3、本文探索性地研究了BPA影響人胚胎早期發(fā)育的表達(dá)基因組學(xué)改變。雙酚A作用于擬胚體，有128個(gè)基因表達(dá)上調(diào)，431個(gè)基因表達(dá)下調(diào)，涉及細(xì)胞分化、分子功能、胚層發(fā)育及信號(hào)傳導(dǎo)通路等細(xì)胞功能和細(xì)胞事件，如PTN和FABP7與神經(jīng)系統(tǒng)發(fā)育相關(guān)。 4、本文僅為系列研究的一部分，，但初步的實(shí)驗(yàn)為課題組今后擴(kuò)大樣本量的實(shí)驗(yàn)、增加驗(yàn)證基因的實(shí)驗(yàn)、設(shè)計(jì)更合適的BPA處理濃度和處理時(shí)間、目標(biāo)基因的功能研究、BPA對(duì)胚胎早期發(fā)育表觀遺傳學(xué)影響等研究工作奠定了基礎(chǔ)。
[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 .

2 銆

本文編號(hào)：2021097