白内障疾病模型小鼠胚胎干细胞建系研究

发布时间：2018-01-19 05:27

本文关键词： 遗传性白内障胚胎干细胞建系 2i培养体系　出处：《浙江理工大学》2012年硕士论文　论文类型：学位论文

【摘要】：白内障是严重影响公众健康的重大疾病。据世界卫生组织统计，全世界约有4500万人失明，其中有一半是由白内障导致的。白内障的遗传方式包括常染色体显性遗传、常染色体隐性遗传和X连锁隐性遗传。目前，科学家主要通过动物模型来研究白内障的发病机理。迄今为止,国内外已发现的遗传性白内障小鼠模型约有140多个，，这些小鼠模型主要是通过自发突变、诱发突变、敲除突变或转基因等方式获得。但关于白内障疾病模型小鼠胚胎干细胞建系的研究目前还未见报道。胚胎干细胞（embryonic stem cells, ESCs）来源于着床前囊胚内细胞团（inner cell mass, ICM）或原始生殖细胞（primordial germ cells，PGCs），是一类具有自我更新和高度分化潜能的细胞。自1981年Evans和Kaufman等人首次成功分离建系小鼠胚胎干细胞以来，人们对胚胎干细胞进行了深入的研究。ES细胞的全能性、无限扩增能力及遗传可操作性使其在早期胚胎发育、转基因动物、人类疾病的发病机理研究、药物筛选、动物疾病模型建立、细胞组织和器官的修复和移植治疗上有着极其诱人的应用前景。本实验以遗传性BALB/cCat/Cat白内障小鼠这一疾病动物模型为研究对象，应用基于“胚胎干细胞自我更新基态”全新理论的2i新型培养体系，即在N2B27基础培养液中添加小分子CHIR99021（GSK3抑制剂）和PD0325901（ERK抑制剂），对来源于3.5dpc的白内障小鼠囊胚的ICM进行培养，从而获得遗传性BALB/cCat/Cat白内障小鼠胚胎干细胞系。经鉴定，该BALB/cCat/CatES细胞以单层或多层密集堆积形成岛状或巢状群体生长，且正常核型率达80%，具高碱性磷酸酶活性；免疫荧光、反转录PCR、Western Blot、实时定量PCR检测显示其表达ES细胞表面特异性抗原SSEA1及多潜能性转录因子Oct4、Nanog、Sox2、Rex1和胚胎干细胞标志基因Eras、Esg、Fgf4、Ulf1、Cripto、Rex1、Gdf3等；拟胚体实验和畸胎瘤实验证实BALB/cCat/CatES在体内外均具有向三个胚层分化的能力。最后，通过胚胎聚合方法获得了4只毛色嵌合的子代小鼠，其中3只患先天性白内障，晶状体出现白色浑浊。先天性白内障是导致儿童视觉异常一个很重要的原因，每10,000个新生婴儿中就可能出现1～6个白内障患者。该白内障疾病模型小鼠胚胎干细胞系的成功建立，填补了白内障动物疾病模型胚胎干细胞领域的空白，为深入研究先天性白内障发病机理和基因治疗单纯性遗传性白内障奠定了基础。
[Abstract]:Cataract is a major disease that seriously affects public health. According to the World Health Organization, about 45 million people worldwide are blind. Half of them are caused by cataract. The inheritance of cataract includes autosomal dominant inheritance, autosomal recessive inheritance and X-linked recessive inheritance. Up to now, there are more than 140 mouse models of hereditary cataract found at home and abroad, which are mainly by spontaneous mutation. Induced mutations, knockout mutations or transgenes were obtained. However, researches on embryonic stem cell lines in cataract model mice have not been reported. Embryonic stem cells (ESCs) originated from inner cell mass of blastocyst before implantation. ICM) or primordial germ cells (PGCs). Since 1981, Evans and Kaufman et al have successfully isolated mouse embryonic stem cells. The totipotency of es cells, the unlimited amplification ability and genetic maneuverability of es cells have been studied in the early embryonic development, transgenic animals and human diseases. Drug screening, animal disease model establishment, cell tissue and organ repair and transplantation therapy have very attractive application prospects. The animal model of hereditary BALB/cCat/Cat cataract in mice was used in this study. A new 2i culture system was applied based on the new theory of "embryonic stem cell self-renewal ground state". That is to say, small molecule CHIR99021(GSK3 inhibitor and PD0325901(ERK inhibitor were added to N2B27 basic culture medium. The ICM of mouse blastocyst derived from 3.5dpc was cultured to obtain the inherited BALB/cCat/Cat cataract mouse embryonic stem cell line. The BALB/cCat/CatES cells were formed into island or nesting population by monolayer or multilayer dense accumulation, and the normal karyotype rate was 80%, and the activity of alkaline phosphatase (ALP) was high. Immunofluorescence, reverse transcription PCR, Western blotanalysis and real-time quantitative PCR analysis showed the expression of es cell surface specific antigen (SSEA1) and multipotent transcription factor (Oct4). Nanogus Sox2 (Rex1) and embryonic stem cell marker gene Erasmor Esgf4Fgf4 Ulf1CriptoRex1 (Gdf3); Embryoid and teratoma experiments confirmed that BALB/cCat/CatES had the ability to differentiate into three embryo layers in vivo and in vitro. Finally. Four hairy chimeric progenies were obtained by the method of embryo aggregation. Three of them suffered from congenital cataract and white opacification of lens. Congenital cataracts are an important cause of visual abnormalities in children, every 10. There may be 1 to 6 cataract patients in 1000 newborn infants. The successful establishment of embryonic stem cell lines in the mouse model of cataract disease has filled the blank in the field of embryonic stem cells of cataract animal disease model. It lays a foundation for studying the pathogenesis of congenital cataract and gene therapy for simple hereditary cataract.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R776.1;R-332

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