人胚胎干细胞体内分化途径获取神经干细胞及DNA甲基化酶Dnmt3a和Dnmt3b表达下调对人胚胎干细胞的影响

发布时间：2018-03-09 11:10

  本文选题：人胚胎干细胞　切入点：神经干细胞　出处：《中南大学》2008年博士论文　论文类型：学位论文

【摘要】： 人类胚胎干细胞(human embryonic stem cells,hESCs)来源于早期胚胎、具有自我更新和分化发育为三个胚层组织潜能的多能性细胞。hESCs在多个领域,如细胞治疗、组织工程、发育生物学、基因功能研究、药物筛选等领域展示了巨大的应用前景。 第一章:人胚胎干细胞株HSF6培养。 目的:探索和建立人胚胎干细胞株HSF6的培养方法。 方法:从ICR胎鼠(E13.5)中分离胚胎成纤维细胞(mouse embryonicfibroblast,MEF),检测丝裂霉素C处理或γ射线照射后MEF的生长状态并作为hESCs饲养层;将hESCs培养于含10ng/mL碱性成纤维细胞生长因子的KO-DMEM培养基中,采用hESCs的酶消化法传代和机械法(巴氏德管制作的传代工具)传代;并对培养的HSF6进行碱性磷酸酶染色、表面标记检测、体外拟胚体(embryoid body,EB)分化能力检测等特征鉴定。 结果:第3-5代的MEF经10μg/mL丝裂霉素C处理1-3小时或经3000Radγ射线照射后能抑制增殖。酶消化法传代后hESCs克隆大小不均,分化克隆容易残留和扩增;机械法传代的hESCs克隆大小较均匀,分化克隆残留较少或培养中容易被剔除,但机械法传代过程繁琐、操作时间较长、工作量大;培养的HSF6能维持未分化状态。 结论:①建立了ICR胎鼠(E13.5)MEF的分离和培养方法,10μg/mL丝裂霉素C处理1-3小时,或3000Radγ射线照射后可作为HSF6的饲养层;②酶消化法和机械法均可用于hESCs的传代。 第二章:hESCs体内分化途径获取神经干细胞。 目的:从hESCs畸胎瘤中分离人神经干细胞(neural progenitor cells,NPC)。 方法:将hESCs注射到SCID鼠体内分化为畸胎瘤,采用贴壁培养法从中分离人NPC;免疫组织化学法检测NPC标记—巢蛋白(nestin);检测NPC分化能力,对分化细胞检测神经元标记—β微管蛋白Ⅲ(Neuron-specific classⅢbeta-tubulin,TuJⅠ)和胶质细胞标记—胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)。 结果:hESCs注射SCID鼠后5-8周后可分化为畸胎瘤,经过贴壁培养和连续传代从中分离到NPC,免疫组织化学检测nestin呈阳性,能分化为神经元和神经胶质细胞,分别表达TuJⅠ和GFAP。 结论:①利用贴壁培养法可从hESCs畸胎瘤中分离到NPC;②“hESCs-SCID鼠-畸胎瘤”模型为细胞组织工程、发育分化研究提供了一个新的方法。 第三章:Dnmt3a和Dnmt3b表达下调对hESCs的影响。 目的:研究DNA新生甲基化酶Dnmt3a(DNA methyltransferase 3a)和Dnmt3b(DNA methyltransferase 3b)表达下调后对hESCs的影响。 方法:观察Dnmt3a与Dnmt3b表达下调后hESCs的生长特性;免疫荧光检测hESCs表面标记;RT-PCR检测维持胚胎干细胞自我更新和维持未分化的相关基因;检测hESCs体外EB分化情况,在不同分化时间行AKP染色和RT-PCR检测三个胚层基因表达情况;检测hESCs在SCID鼠体内分化能力。 结果:Dnmt3a和Dnmt3b表达下调后hESCs:在MEF上呈克隆式生长,表面标记检测呈hESC特征,表达Oct3/4、Sox2和Nanog;EB分化障碍、AKP消退延迟,Dnmt3b下调后hESCs分化时神经外胚层标记基因提前出现;Dnmt3a和Dnmt3b表达下调后hESCs在SCID小鼠体内未形成畸胎瘤。 结论:①Dnmt3a和Dnmt3b表达下调后不影响hESCs未分化状态的维持,推测Dnmt3a和Dnmt3b不是hESCs未分化状态维持所必需的;②Dnmt3a和Dnmt3b表达下调导致hESCs分化缺陷,推测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.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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