【作者】 曾桥；

【导师】 张灼华； 夏昆； 王丹玲；

【作者基本信息】 中南大学 ， 遗传学， 2008， 博士

【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。更多还原

【Abstract】 Human embryonic stem cells(hESCs) were derived from human early blastocysts,were able to be expanded long-term in vitro with stable karyotype,and were able to self-renewal and differentiate into all cells types of all three germ layers.The hESCs provided great promise for regenerative medicine and provided valuable tools for researching early human embryonic development and differentiation,gene functions and for evaluating of pharmacologic action.Based on introducing hESCs stain-HSF6,following studies were carried out.Chapter 1 Study on culturing system of hESCs-HSF6.Aims:to study and establish culturing system of hESCs-HSF6.Methods:MEF cells were derivated from embryos of 13.5-days-pregency ICR mice.The MEF were treated with 10μg/mL mitomycin C or 3 000 Radγ-ray.The hESCs were maintained on the irradiated MEF in KO-DMEM with daily medium changes.The hESCs were passaged using two methods:enzymatical method with 1mg/mL collagenaseⅣand lmg/mL dispase and machanical method with Pasteur glass pipets.The characterizations of hESCs were carried out using immunostaining and differentiating.Results:the passage 3 to 5 MEF did not proliferate after being treated by 10μg/mL mitomycin C for 1-3 hours or 3 000 Radγ-ray.The enzyme-treated expansion rapidly produced greater amounts of hESCs within a limited time frame.However,the cell clumps varied in size,and there was a probability that both the differentiated and undifferentiated cells would be transferred.The mechanical method was laborious and time-consuming,but the technique permitted efficient transfer of undifferentiated hESCs and results in similar clump sizes.In cases in which there were differentiated colonies,the combination of two methods allows mass production of hESCs by excluding differentiated colonies from passage by manual selection before enzyme treatment.Results of immunostaining and differentiaon showed that the hESCs could self-renewal and maintain undifferetiation in the culture system.Conclusions:①ICR-MEF were derivated successfully from embryos of 13.5-days-pregency ICR mice;it was efficient to make feeder for hESCs-HSF6 with passage 3 to 5 ICR-MEF treated by 10μg/mL mitomycin C for 1-3 hours or 3 000 Radγ-ray MEF;②Both mechanical and enzymatic transfer methods for hESCs depent on experimental purpose. The mechanical transfer method was good for maintenance of hESC lines.Chapter 2 Derivation of NPC from teratomas of hESCs.Aims:to differentiate hESCs into teratomas in vivo and to derivate neural progenitor cells(NPC) from the teratomas.Methods:hESCs clumps were harvested and were injected into SCID-beige mice.The mice were sacrificed and teratomas were got and identified.NPC were derivated from teratomas through plating cells onto polyornithine-(PO) and fibronectin(FN)-coated dishes in serum-free medium DMEM/F12 supplemented with B27 supplement,and 10ng/ml bFGF.The NPC were fixed for immunostaining with mouse monoclonal anti-nestin.The NPC were induced to differentiate by withdrawing bFGF and the differenctiated cells were fixed for immunostaining with mouse monoclonal anti-TuJⅠand mouse monoclonal anti-GFAP.Results:The hESCs grafted into SCID mice consistently developed into teratomas after 5- to 8-week.They were analyzed histologically and contained representative tissues of the three germ layers.NPCs were derived successfully from the teratomas of hESCs through consecutive passages and culture in PO/FN-coated plate.Using antibodies against neural stem cell marker nestin,the cells were detected nestin positive.The NPC could differentiate into neurons and astrocytes.Using antibodies against neuronal markerβⅢtubulin(TuJⅠantigen) and astroglial marker glial fibrillary acidic protein(GFAP),it was found that TuJⅠ-positive or GFAP-positive cells were presented in these differentiated teratoma-derived NPCs.Conclusions:①NPCs were derived successfully from the teratomas of hESCs through culture in PO/FN-coated plate;②the model of "hESCs-SCID mice-teratoma" provided a new approach or tool for cell-based therapy and reseach of differentiation or development.Chapter 3 Effects of knockdown of Dnmt3a and Dnmt3b on hESCs.Aims:to study on effects of knockdown of DNA methyltransferase 3a (Dnmt3a) and DNA methyltransferase 3b(Dnmt3b) on hESCs.Methods:The morphous and growth of hESCs were observed after the Dnmt3a and Dnmt3b were knockdown.Immunostaining with makers of hESCs were used to detect their characterizations.Reverse transcription PCR(RT-PCR) was used to detect Oct3/4,Nanog and Sox2 that were correlated to hESCs self-renewal.Potentials of differentiation of hESCs after being knockdown Dnmt3a and Dnmt3b were examined:in vitro be induced into embryoid body(EB) and in vivo be induced into teratoma.Results:The hESCs could maintain self-renewal and undifferentiation after being knockdown Dnmt3a and Dnmt3b,and they grow colonly on MEF similar to wild type hESCs.Results of immunostaining showed that they still were SSEA-4-,TRA-1-60-,TRA-1-81- and OCT3/4-positive. They still expressed Oct3/4,Nanog and Sox2.The AKP staining of EB showd that they were difficult to differentiate into embryo bodies(EB) in vitro and knockdown of Dnmt3b could cause neuroectoderm emerge ahead of schedule in EBs.Both hESCs of knockdown of Dnmt3a and Dnmt3b could not differentiate into teratoma in SCID mice.Conclusions:①Dnmt3a and Dnmt3b may be not essential for hESC maintaining self-renewal and undifferentiation;②Dnmt3a and Dnmt3b may be essential for hESC differentiation normally.更多还原