本文關(guān)鍵詞： 遺傳性白內(nèi)障 胚胎干細(xì)胞 建系 2i培養(yǎng)體系　出處：《浙江理工大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：白內(nèi)障是嚴(yán)重影響公眾健康的重大疾病。據(jù)世界衛(wèi)生組織統(tǒng)計(jì)，全世界約有4500萬人失明，其中有一半是由白內(nèi)障導(dǎo)致的。白內(nèi)障的遺傳方式包括常染色體顯性遺傳、常染色體隱性遺傳和X連鎖隱性遺傳。目前，科學(xué)家主要通過動物模型來研究白內(nèi)障的發(fā)病機(jī)理。迄今為止,國內(nèi)外已發(fā)現(xiàn)的遺傳性白內(nèi)障小鼠模型約有140多個，，這些小鼠模型主要是通過自發(fā)突變、誘發(fā)突變、敲除突變或轉(zhuǎn)基因等方式獲得。但關(guān)于白內(nèi)障疾病模型小鼠胚胎干細(xì)胞建系的研究目前還未見報(bào)道。 胚胎干細(xì)胞（embryonic stem cells, ESCs）來源于著床前囊胚內(nèi)細(xì)胞團(tuán)（inner cell mass, ICM）或原始生殖細(xì)胞（primordial germ cells，PGCs），是一類具有自我更新和高度分化潛能的細(xì)胞。自1981年Evans和Kaufman等人首次成功分離建系小鼠胚胎干細(xì)胞以來，人們對胚胎干細(xì)胞進(jìn)行了深入的研究。ES細(xì)胞的全能性、無限擴(kuò)增能力及遺傳可操作性使其在早期胚胎發(fā)育、轉(zhuǎn)基因動物、人類疾病的發(fā)病機(jī)理研究、藥物篩選、動物疾病模型建立、細(xì)胞組織和器官的修復(fù)和移植治療上有著極其誘人的應(yīng)用前景。 本實(shí)驗(yàn)以遺傳性BALB/cCat/Cat白內(nèi)障小鼠這一疾病動物模型為研究對象，應(yīng)用基于“胚胎干細(xì)胞自我更新基態(tài)”全新理論的2i新型培養(yǎng)體系，即在N2B27基礎(chǔ)培養(yǎng)液中添加小分子CHIR99021（GSK3抑制劑）和PD0325901（ERK抑制劑），對來源于3.5dpc的白內(nèi)障小鼠囊胚的ICM進(jìn)行培養(yǎng)，從而獲得遺傳性BALB/cCat/Cat白內(nèi)障小鼠胚胎干細(xì)胞系。經(jīng)鑒定，該BALB/cCat/CatES細(xì)胞以單層或多層密集堆積形成島狀或巢狀群體生長，且正常核型率達(dá)80%，具高堿性磷酸酶活性；免疫熒光、反轉(zhuǎn)錄PCR、Western Blot、實(shí)時(shí)定量PCR檢測顯示其表達(dá)ES細(xì)胞表面特異性抗原SSEA1及多潛能性轉(zhuǎn)錄因子Oct4、Nanog、Sox2、Rex1和胚胎干細(xì)胞標(biāo)志基因Eras、Esg、Fgf4、Ulf1、Cripto、Rex1、Gdf3等；擬胚體實(shí)驗(yàn)和畸胎瘤實(shí)驗(yàn)證實(shí)BALB/cCat/CatES在體內(nèi)外均具有向三個胚層分化的能力。最后，通過胚胎聚合方法獲得了4只毛色嵌合的子代小鼠，其中3只患先天性白內(nèi)障，晶狀體出現(xiàn)白色渾濁。 先天性白內(nèi)障是導(dǎo)致兒童視覺異常一個很重要的原因，每10,000個新生嬰兒中就可能出現(xiàn)1～6個白內(nèi)障患者。該白內(nèi)障疾病模型小鼠胚胎干細(xì)胞系的成功建立，填補(bǔ)了白內(nèi)障動物疾病模型胚胎干細(xì)胞領(lǐng)域的空白，為深入研究先天性白內(nèi)障發(fā)病機(jī)理和基因治療單純性遺傳性白內(nèi)障奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R776.1;R-332

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相關(guān)期刊論文 前3條

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