本文選題：人胚胎干細(xì)胞　切入點(diǎn)：神經(jīng)干細(xì)胞　出處：《中南大學(xué)》2008年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 人類(lèi)胚胎干細(xì)胞(human embryonic stem cells,hESCs)來(lái)源于早期胚胎、具有自我更新和分化發(fā)育為三個(gè)胚層組織潛能的多能性細(xì)胞。hESCs在多個(gè)領(lǐng)域,如細(xì)胞治療、組織工程、發(fā)育生物學(xué)、基因功能研究、藥物篩選等領(lǐng)域展示了巨大的應(yīng)用前景。 第一章:人胚胎干細(xì)胞株HSF6培養(yǎng)。 目的:探索和建立人胚胎干細(xì)胞株HSF6的培養(yǎng)方法。 方法:從ICR胎鼠(E13.5)中分離胚胎成纖維細(xì)胞(mouse embryonicfibroblast,MEF),檢測(cè)絲裂霉素C處理或γ射線(xiàn)照射后MEF的生長(zhǎng)狀態(tài)并作為hESCs飼養(yǎng)層;將hESCs培養(yǎng)于含10ng/mL堿性成纖維細(xì)胞生長(zhǎng)因子的KO-DMEM培養(yǎng)基中,采用hESCs的酶消化法傳代和機(jī)械法(巴氏德管制作的傳代工具)傳代;并對(duì)培養(yǎng)的HSF6進(jìn)行堿性磷酸酶染色、表面標(biāo)記檢測(cè)、體外擬胚體(embryoid body,EB)分化能力檢測(cè)等特征鑒定。 結(jié)果:第3-5代的MEF經(jīng)10μg/mL絲裂霉素C處理1-3小時(shí)或經(jīng)3000Radγ射線(xiàn)照射后能抑制增殖。酶消化法傳代后hESCs克隆大小不均,分化克隆容易殘留和擴(kuò)增;機(jī)械法傳代的hESCs克隆大小較均勻,分化克隆殘留較少或培養(yǎng)中容易被剔除,但機(jī)械法傳代過(guò)程繁瑣、操作時(shí)間較長(zhǎng)、工作量大;培養(yǎng)的HSF6能維持未分化狀態(tài)。 結(jié)論:①建立了ICR胎鼠(E13.5)MEF的分離和培養(yǎng)方法,10μg/mL絲裂霉素C處理1-3小時(shí),或3000Radγ射線(xiàn)照射后可作為HSF6的飼養(yǎng)層;②酶消化法和機(jī)械法均可用于hESCs的傳代。 第二章:hESCs體內(nèi)分化途徑獲取神經(jīng)干細(xì)胞。 目的:從hESCs畸胎瘤中分離人神經(jīng)干細(xì)胞(neural progenitor cells,NPC)。 方法:將hESCs注射到SCID鼠體內(nèi)分化為畸胎瘤,采用貼壁培養(yǎng)法從中分離人NPC;免疫組織化學(xué)法檢測(cè)NPC標(biāo)記—巢蛋白(nestin);檢測(cè)NPC分化能力,對(duì)分化細(xì)胞檢測(cè)神經(jīng)元標(biāo)記—β微管蛋白Ⅲ(Neuron-specific classⅢbeta-tubulin,TuJⅠ)和膠質(zhì)細(xì)胞標(biāo)記—膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein,GFAP)。 結(jié)果:hESCs注射SCID鼠后5-8周后可分化為畸胎瘤,經(jīng)過(guò)貼壁培養(yǎng)和連續(xù)傳代從中分離到NPC,免疫組織化學(xué)檢測(cè)nestin呈陽(yáng)性,能分化為神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞,分別表達(dá)TuJⅠ和GFAP。 結(jié)論:①利用貼壁培養(yǎng)法可從hESCs畸胎瘤中分離到NPC;②“hESCs-SCID鼠-畸胎瘤”模型為細(xì)胞組織工程、發(fā)育分化研究提供了一個(gè)新的方法。 第三章:Dnmt3a和Dnmt3b表達(dá)下調(diào)對(duì)hESCs的影響。 目的:研究DNA新生甲基化酶Dnmt3a(DNA methyltransferase 3a)和Dnmt3b(DNA methyltransferase 3b)表達(dá)下調(diào)后對(duì)hESCs的影響。 方法:觀察Dnmt3a與Dnmt3b表達(dá)下調(diào)后hESCs的生長(zhǎng)特性;免疫熒光檢測(cè)hESCs表面標(biāo)記;RT-PCR檢測(cè)維持胚胎干細(xì)胞自我更新和維持未分化的相關(guān)基因;檢測(cè)hESCs體外EB分化情況,在不同分化時(shí)間行AKP染色和RT-PCR檢測(cè)三個(gè)胚層基因表達(dá)情況;檢測(cè)hESCs在SCID鼠體內(nèi)分化能力。 結(jié)果:Dnmt3a和Dnmt3b表達(dá)下調(diào)后hESCs:在MEF上呈克隆式生長(zhǎng),表面標(biāo)記檢測(cè)呈hESC特征,表達(dá)Oct3/4、Sox2和Nanog;EB分化障礙、AKP消退延遲,Dnmt3b下調(diào)后hESCs分化時(shí)神經(jīng)外胚層標(biāo)記基因提前出現(xiàn);Dnmt3a和Dnmt3b表達(dá)下調(diào)后hESCs在SCID小鼠體內(nèi)未形成畸胎瘤。 結(jié)論:①Dnmt3a和Dnmt3b表達(dá)下調(diào)后不影響hESCs未分化狀態(tài)的維持,推測(cè)Dnmt3a和Dnmt3b不是hESCs未分化狀態(tài)維持所必需的;②Dnmt3a和Dnmt3b表達(dá)下調(diào)導(dǎo)致hESCs分化缺陷,推測(cè)Dnmt3a和Dnmt3b是hESCs正常分化所必需的。
[Abstract]:Human embryonic stem cells (human embryonic stem cells, hESCs) derived from the early embryo, self-renewal and differentiation of three germ layers potential of pluripotent cells.HESCs in many fields, such as cell therapy, tissue engineering, developmental biology, gene function research, drug screening and other areas show great application prospect.
Chapter 1: HSF6 culture of human embryonic stem cell line.
Objective: To explore and establish the culture method of human embryonic stem cell line HSF6.
Methods: from ICR fetal rats (E13.5) isolated from embryonic fibroblast cells (mouse, embryonicfibroblast, MEF) detection of growth state of mitomycin C treatment or after irradiation, MEF and hESCs as feeder layer; hESCs were cultured in 10ng/mL containing basic fibroblast growth factor KO-DMEM medium by enzyme digestion. And the mechanical method of hESCs (Pap de tube produced by the passage passage; and the tool) HSF6 cultured by alkaline phosphatase staining, surface markers, in vitro embryoid bodies (embryoid body, EB) differentiation detection characteristics identified.
Results: the 3-5 generation of MEF by 10 g/mL mitomycin C treatment for 1-3 h or 3000Rad after gamma irradiation can inhibit the proliferation of enzyme digestion. After passage of hESCs clone size is not uniform, and easy to clone differentiation residue amplification; mechanical cutting hESCs clones with uniform sizes, residual or less differentiation cloning culture easily eliminated, but mechanical cutting process cumbersome, longer operation time, heavy workload; cultured HSF6 can maintain undifferentiated state.
Conclusion: (1) a method of isolation and culture of ICR fetal rat (E13.5) MEF was established. After 10 hours of mitomycin C treatment for 1-3 hours, or after 3000Rad gamma ray irradiation, it can serve as a feeder layer for HSF6. Second, enzyme digestion and mechanical method can be used for hESCs generation. MEF
The second chapter: neural stem cells are obtained by the differentiation pathway of hESCs in vivo.
Objective: to isolate human neural stem cells (neural progenitor cells, NPC) from hESCs teratoma.
Methods: hESCs was injected into SCID mice for differentiation of teratoma by adherent culture method isolated from human NPC; detection of NPC marker nestin immunohistochemical method (nestin); detection of NPC differentiation ability of differentiated cells to detect neuronal marker beta tubulin III (Neuron-specific III class beta-tubulin TuJ 1) and glial cell marker glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP).
Results: hESCs could be differentiated into teratoma after 5-8 weeks of SCID injection, and NPC was isolated from adherent culture and continuous passage. Nestin was positive by immunohistochemistry, and could differentiate into neurons and glial cells, expressing TuJ I and GFAP. respectively.
Conclusion: 1. NPC can be isolated from hESCs teratoma by adherent culture. 2. HESCs-SCID mouse teratoma model provides a new method for cell tissue engineering and developmental differentiation.
The third chapter: the effect of down regulation of Dnmt3a and Dnmt3b on hESCs.
Objective: To study the effect of down-regulation of DNA new methylation enzyme Dnmt3a (DNA methyltransferase 3a) and Dnmt3b (DNA methyltransferase 3b) on hESCs.
Methods: To observe the Dnmt3a expression of Dnmt3b and growth characteristics of hESCs by immunofluorescence; hESCs surface marker; RT-PCR gene detection of embryonic stem cell self-renewal and differentiation; detection of hESCs EB in vitro differentiation, the expression in the three embryonic genes of different differentiation time by AKP staining and RT-PCR assay in detection of hESCs; SCID in vivo differentiation ability.
Results: Dnmt3a and Dnmt3b expression after hESCs: MEF was cloned in growth, surface marker detection showed hESC features, expression of Oct3/4, Sox2 and Nanog; EB differentiation disorder, AKP regression delay, neuroectodermal marker genes occur early downregulation of Dnmt3b after hESCs differentiation; Dnmt3a and down-regulation of Dnmt3b hESCs in SCID mice is not formed teratoma.
Conclusion: the expression of Dnmt3a and Dnmt3b maintain down hESCs does not affect the undifferentiated state, suggesting that Dnmt3a and Dnmt3b are not required to maintain the undifferentiated state of hESCs; the expression of Dnmt3a and Dnmt3b resulted in the downregulation of hESCs differentiation defects, suggesting that Dnmt3a and Dnmt3b are required for the normal differentiation of hESCs.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R329

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