【摘要】：大鼠是一種非常有價值的研究病理和藥理的模型動物。因此建立一種穩(wěn)定的大鼠胚胎干細(xì)胞(rES)分化體系對于分析rES早期發(fā)育、基因功能以及應(yīng)用于基因打靶制作人類疾病動物模型非常必要。盡管具有生殖系遺傳能力的rES細(xì)胞系已經(jīng)被成功建立,維持其自我更新和保持其未分化的狀態(tài)的各種分子網(wǎng)絡(luò)已經(jīng)被很好的揭示,但是,rES細(xì)胞向三胚層分化體系和方法的相關(guān)研究甚少。中樞神經(jīng)系統(tǒng)退行性疾病嚴(yán)重危害著人類的健康,ES細(xì)胞有希望為神經(jīng)系統(tǒng)疾病細(xì)胞替代性治療提供細(xì)胞來源。到目前為止,尚無關(guān)于自體rES細(xì)胞獲得神經(jīng)干細(xì)胞(NS)的報道。研究rES細(xì)胞的神經(jīng)分化過程,建立從全能性細(xì)胞向神經(jīng)細(xì)胞誘導(dǎo)分化過程,獲得具有NS細(xì)胞特性的神經(jīng)前體細(xì)胞,不僅為疾病細(xì)胞治療的實驗研究提供理論基礎(chǔ),而且為神經(jīng)系統(tǒng)早期發(fā)育的研究提供良好的模型。此研究擬通過建立rES細(xì)胞系,對其建立穩(wěn)定的體外分化體系,進而誘導(dǎo)分化為具有自我更新和多項分化潛能的NS細(xì)胞。希望能為大鼠神經(jīng)系統(tǒng)發(fā)育過程的研究平臺,藥理檢測及神經(jīng)疾病模型的建立提供理論依據(jù)。經(jīng)過研究得到以下實驗結(jié)果： 1)本研究取孕E14.5DA大鼠胚胎腦組織細(xì)胞,在體外無血清條件下(N2B27添加bFGF和mEGF生長因子)進行培養(yǎng),獲取可以以單層貼壁細(xì)胞和懸浮神經(jīng)球的狀態(tài)進行傳代培養(yǎng)rNS。細(xì)胞免疫熒光檢測結(jié)果顯示NS細(xì)胞表面標(biāo)志Nestin、Olig2、Pax6、呈陽性；大部分rNS表達MKi-67,證明rNS處于增殖旺盛的狀態(tài)。神經(jīng)元祖細(xì)胞的表面標(biāo)志Dcx的表達率為0.8%,初步說明其有能夠向神經(jīng)元分化的潛能。 2)利用rNS細(xì)胞分化培養(yǎng)基對rNS進行體外分化,10d左右,細(xì)胞免疫熒光結(jié)果顯示三種神經(jīng)細(xì)胞系的表面標(biāo)志Tuj1、GFAP和04的表達為陽性,說明rNS細(xì)胞能夠分化為神經(jīng)元,星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞。對其進一步進行分化,進行細(xì)胞免疫熒光檢測發(fā)現(xiàn)神經(jīng)元亞型標(biāo)志物MAP2、GABA和ChAT表達為陽性,說明此rNS細(xì)胞具有向不同神經(jīng)元亞型分化的能力。綜合以上兩點初步確定我們從大鼠腦組織分離培養(yǎng)的細(xì)胞為NS細(xì)胞。 3)利用2i體系的rES細(xì)胞培養(yǎng)基進行培養(yǎng),獲得10個穩(wěn)定傳代的rES細(xì)胞系,共培養(yǎng)了30代,沒有觀察到明顯的分化跡象。對DA5-3rES細(xì)胞進行了堿性磷酸酶染色(APStaining)發(fā)現(xiàn)為AP呈陽性,RT-PCR檢測進一步發(fā)現(xiàn)DA5-3rES表達Oct4、Sox2、Nanog、Rex-1、fgf4和Eras多能性基因。細(xì)胞免疫熒光染色結(jié)果表明DA5-3rES細(xì)胞表達Oct4、Sox2和SSEA-1多能性因子,核型分析結(jié)果顯示,有超過75%的細(xì)胞染色體條數(shù)為42,說明這株DA5-3rES細(xì)胞具有自我更新和多向分化的潛能且核型正常。 4)rES在正常含有10%血清(DM)的EB培養(yǎng)基條件下,大部分rES細(xì)胞發(fā)生凋亡或者壞死,只能形成少數(shù)EB(38.37±4.4%)；利用階段性去除2i的誘導(dǎo)方式,rES在rEFM,CM+2i,及CM的培養(yǎng)條件下順次分別培養(yǎng)2d,2d,1d,能夠形成形態(tài)均一,質(zhì)量良好的EB,說明此rEB誘導(dǎo)體系穩(wěn)定可行。 5)利用RT-PCR技術(shù)檢測rEB體外分化的基因表達情況, rEB表達外、中、內(nèi)三胚層的表面標(biāo)志基因Nestin、Sox17、Afp、Flk和Gata6; rEB貼壁分化的過程中,第6-15d,能夠分化為呈規(guī)律性搏動的心肌細(xì)胞。由此初步確定rEB具有向三胚層體外分化的能力。 6)利用N2B27培養(yǎng)基對rEB進行體外神經(jīng)干細(xì)胞的分化,在誘導(dǎo)第11d左右形成神經(jīng)管上皮細(xì)胞,挑取克隆進行貼壁培養(yǎng),形成rNS細(xì)胞,細(xì)胞免疫熒光結(jié)果顯示Nestin、 Olig2、Pax6、Sox2呈陽性,大部分rNS細(xì)胞表達MKi-67證明rNS細(xì)胞處于增殖旺盛的狀態(tài)。神經(jīng)元祖細(xì)胞的表面標(biāo)志Dcx的表達率為0.6%,初步說明其有能夠向神經(jīng)元分化的潛能。 7)通過使用rNS細(xì)胞分化培養(yǎng)基對rNS進行體外分化,10d左右,細(xì)胞免疫熒光檢測結(jié)果顯示三種神經(jīng)細(xì)胞系的表面標(biāo)志Tuj1、GFAP和04的表達為陽性,說明rNS能夠分化為神經(jīng)元,星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞。對其進一步進行分化,檢測發(fā)現(xiàn)神經(jīng)元亞型標(biāo)志物MAP2、TH和GABA表達為陽性,說明此rNS具有向不同神經(jīng)元亞型分化的能力。 綜合以上,DA5-3rES體外分化為神經(jīng)干細(xì)胞體系的建立具有可行性和有效性。
[Abstract]:Rats are a very valuable model animal for the study of pathology and pharmacology. Therefore, it is necessary to establish a stable rat embryonic stem cell (rES) differentiation system for the analysis of the early development of rES, the gene function and the application of the gene targeting to the production of human disease animal models. Although the rES cell line with a reproductive system genetic capacity has been successfully established, various molecular networks that maintain its self-renewal and maintain its undifferentiated state have been well disclosed, but the correlation of the rES cell to the three-germ differentiation system and method is very small. The degenerative disease of the central nervous system is a serious threat to human health, and ES cells are expected to provide a source of cell for alternative treatment of nervous system disease cells. To date, there is no report on autologous rES cells to obtain neural stem cells (NS). To study the process of neural differentiation of rES cells, the process of inducing differentiation from functional cells to nerve cells was established, and the neural precursor cells with NS cell characteristics were obtained, which not only provided a theoretical basis for the experimental study of disease cell therapy, But also provides a good model for the early development of the nervous system. This study is to establish a stable in vitro differentiation system to induce differentiation into NS cells with self-renewal and multiple differentiation potential by establishing a rES cell line. It is desirable to provide a theoretical basis for the establishment of a research platform, a pharmacological test and a neural disease model for the development of the nervous system of the rat. The following experimental results were obtained through the study: 1) In this study, the embryonic brain tissue cells of E14. 5DA rats were cultured and cultured under no serum conditions in vitro (addition of bFGF and mEGF growth factor to N2B27). S. The results showed that the NS cell surface marker, Nestin, Olig2, Pax 6, was positive, most of the rNS expression was Mk-67, and it was proved that the rNS was in the form of vigorous proliferation. The expression rate of the surface marker Dcx of the neuron progenitor cells was 0.8%, indicating that it was able to differentiate into the neurons. (2) The in vitro differentiation of rNS was carried out by using the rNS cell differentiation medium, and the results showed that the expression of the surface markers Tuj1, GFAP and 04 of the three nerve cell lines was positive, indicating that the rNS cells could be differentiated into neurons, astrocytes and oligodendrocytes. It was found that the expression of MAP2, GABA and ChAT of the neuron-type markers was positive, indicating that the rNS cells were differentiated into different neuron subtypes. The ability to separate the cultured cells from the rat brain tissue was preliminarily determined at the above two points. S-cells.3) The rES cell lines of the 2i system were cultured to obtain 10 stable passages of the rES cell line, which was co-cultured for 30 generations and no significant observation was observed. The differentiation of DA5-3rES cells was found to be positive for AP. RT-PCR was used to detect the expression of Oct4, Sox2, Nanog, Rex-1, fgf4, and Eras in DA5-3rES. The results of the cellular immunofluorescent staining show that the DDA5-3rES cells express Oct4, Sox2 and SSEA-1. The results show that the number of the cells with more than 75% of the cells is 42, indicating that the DDA5-3rES cell has the potential of self-renewal and multi-directional differentiation. And the karyotype was normal. (4) in the case of the EB medium containing 10% serum (DM) normally, most of the rES cells had apoptosis or necrosis, only a few of EB (38.37-4.4%) were formed, and the rES was sequentially removed under the conditions of rEFM, CM + 2i and CM under the culture conditions of rEFM, CM + 2i, and CM, respectively. 2 d, 2d and 1d were cultured to form an EB with good morphology and good quality. (5) The expression of rEB in vitro was detected by RT-PCR, and the surface marker genes, Nestin, Sox17, Afp, Flk and Gaata6, were detected by RT-PCR. The primary determination of rEB has a three-phase, three-phase, three-phase, three-dimensional, (6) the differentiation of the in vitro neural stem cells was carried out by using the N2B27 medium, and the neural tube epithelial cells were formed on the left and right of the induction of the 11th day, and the clones were selected for adherent culture to form the rNS cells, and the result of the immunofluorescence of the cells showed that the Nestin, Oli2, P ax6, Sox2 were positive, most of the rNS cells expressed MKi-67 to demonstrate the rNS The expression rate of the surface marker Dcx of the neuron progenitor cells was 0.6%. 7) In vitro differentiation of the rNS by using the rNS cell differentiation medium, the expression of the surface markers Tuj1, GFAP and 04 of the three nerve cell lines is positive, indicating that the rNS can be differentiated into neurons, Astrocytes and oligodendrocytes were further differentiated, and the expression of MAP2, TH and GABA was found to be positive for neuronal subtype markers, indicating that this rNS had In vitro, the differentiation of DA5-3rES into neural stem cells in vitro
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R329

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