人胚胎生殖细胞体外培养体系的建立及其向心肌前体细胞分化的初步研究

发布时间：2018-04-14 11:29

本文选题：人胚胎生殖腺嵴和肠背系膜来源成纤维样细胞 + 人胚胎生殖细胞　；参考：《吉林大学》2007年博士论文

【摘要】： 为优化人胚胎生殖细胞(hEG)的体外培养体系及研究5-氮杂胞苷对hEG向心肌前体细胞分化的影响,我们分离培养了人胚胎生殖腺嵴和肠背系膜中原始生殖细胞(PGCs)周围的成纤维样细胞,证实其具有稳定的生物学特性,并首次证实其表达体外培养hEG所必需的细胞因子。以此人胚胎成纤维样细胞作为饲养层细胞,模拟PGCs在早期胚胎发育过程中不断迁移、扩增并保持未分化状态时的体内环境,同时添加bFGF、LIF和forskolin细胞因子,体外成功培养hEG,并对其生物学特性进行鉴定。所培养的hEG可传8～10代,并能够自我更新,保持未分化状态、正常人类二倍体核型及多向分化能力,建立了一种新的hEG体外培养方法。为hEG的进一步研究及其临床应用奠定了基础。在此基础上,进一步利用半定量逆转录多聚酶链反应检测5-氮杂胞苷对hEG向心肌前体细胞分化的影响。初步研究表明,5-氮杂胞苷可能具有促进hEG向心肌前体细胞分化的能力。同时,我们亦自人早期胚胎中分离培养人胚胎心肌细胞,并对其生物学特性进行初步研究。为今后心血管疾病的干细胞移植治疗研究奠定了基础。
[Abstract]:In order to optimize the culture system of human embryonic germ cells (EGG) in vitro and to study the effect of 5-azacytidine on the differentiation of hEG into cardiomyocyte precursor cells,We isolated and cultured fibroblasts around the primordial germ cells (PGCs) of human embryonic gonadal ridge and dorsal mesenteric membrane, and confirmed their stable biological characteristics, and demonstrated for the first time the expression of cytokines necessary for the culture of hEG in vitro.Human embryonic fibroblast cells were used as feeder layer cells to mimic the migration of PGCs during early embryonic development, to amplify and maintain the undifferentiated environment in vivo, and to add bFGF LIF and forskolin cytokines at the same time.HEGG was successfully cultured in vitro and its biological characteristics were identified.The cultured hEG can be transferred for 810 generations, and can renew itself, maintain undifferentiated state, normal human diploid karyotype and multidirectional differentiation ability. A new method of hEG culture in vitro was established.It lays a foundation for the further study and clinical application of hEG.On this basis, semi-quantitative reverse transcription polymerase chain reaction was used to detect the effect of 5-azacytidine on the differentiation of hEG into cardiomyocyte precursor cells.The preliminary study showed that 5-azacytidine might promote the differentiation of hEG into cardiomyocyte precursor cells.At the same time, human embryonic cardiomyocytes were isolated from human early embryos and their biological characteristics were studied.It will lay a foundation for the research of stem cell transplantation therapy for cardiovascular disease in the future.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329

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