早衰小鼠P1型（SAMP1）胚胎干细胞的建系研究

发布时间：2018-04-09 22:08

本文选题：胚胎干细胞　切入点：SAMP1小鼠　出处：《浙江理工大学》2011年硕士论文

【摘要】：自1981年小鼠胚胎干细胞成功建系以来,人们对胚胎干细胞的研究不断深入:(1)胚胎干细胞已成为研究细胞分化、器官发生以及了解胚胎发育机制的有效手段;(2)对胚胎干细胞进行基因操作有助于确定特定基因在发育、生理和病理过程中的作用;(3)成功建系人类疾病动物模型的胚胎干细胞,结合近年来兴起的基因打靶(基因敲除或基因敲入)等生物技术,可以研究人类高血压、早衰等多种自发性疾病的发生机制,并对其治疗和预防提供科学依据。胚胎干细胞建系是进行所有这些研究的前提和基础。虽然胚胎干细胞建系技术已经获得了不断的丰富和完善,但是二十多年以来能够进行生殖系传代明确建系的种属仍然仅限于小鼠有限的几种品系,LIF加饲养层的有血清培养体系并不能通用于所有的小鼠及其他物种的胚胎干细胞建系工作。2008年华人科学家应其龙博士领头的研究小组基于“胚胎干细胞自我更新基态”理论利用2i小分子培养体系成功建立大鼠胚胎干细胞系为更多的小鼠品系甚至其它物种的胚胎干细胞建系工作带来了全新的理论与技术支持。本实验以医学研究热点之一的老年性疾病的动物实验模型早衰小鼠P1型即SAMP1小鼠作为研究对象,旨在通过应用新型的添加小分子的无血清2i新型培养体系对分离的SAMP1小鼠囊胚的内细胞团(ICM)进行培养而获得胚胎干细胞,成功建立SAMP1小鼠疾病动物模型的胚胎干细胞系。建立该疾病模型SAMP1小鼠胚胎干细胞系具有重大意义(:1)目前国内外从未有过早衰小鼠模型胚胎干细胞成功建系的报道,应用2i小分子培养体系建系成功很好的验证了这种培养体系的通用型;(2)对这种疾病模型小鼠胚胎干细胞的研究有助于人们清楚了解早衰的遗传学特征,从而确定疾病相关基因,并实施基因打靶等基因矫正技术,根据过早衰老、老年性耳聋等相关疾病的发生机理有的放矢,对症下药,为这类疾病最终的治疗和攻克奠定基础。本实验通过分离SAMP1小鼠囊胚内细胞团(ICM)、对其进行培养并对获得的胚胎干细胞进病毒感染,得出: 1. 3.5d SAMP1小鼠囊胚可以在2i培养条件下自然孵化出内细胞团(ICM),进一步培养可获得类胚胎干细胞样克隆; 2.对这些类胚胎干细胞样克隆进行AKP染色,Oct4,Sox2,SSEA-1基因表达免疫荧光染色以及Reverse Transcription PCR等胚胎干细胞的鉴定标准试验,表明这些呈克隆生长的细胞即为胚胎干细胞; 3.用pCDNA-GFP慢病毒感染获得的SAMP1胚胎干细胞,可以得到持久携带绿荧光蛋白基因的SAMP1小鼠胚胎干细胞,传代次数30代以上仍可观察到荧光。
[Abstract]:Since the successful establishment of mouse embryonic stem cells in 1981, the research on embryonic stem cells has gone deeper and deeper.) embryonic stem cells have become the study of cell differentiation.Organogenesis and an effective means of understanding the mechanisms of embryonic development: genetic manipulation of embryonic stem cells helps to determine the role of specific genes in the development, physiology and pathology of embryonic stem cells) successful in the establishment of animal models of human disease,Combined with the biotechnology such as gene targeting (gene knockout or gene knockin) developed in recent years, we can study the mechanism of spontaneous diseases such as human hypertension, premature senility and so on, and provide scientific basis for its treatment and prevention.Embryonic stem cell line building is the prerequisite and basis for all these studies.Although embryonic stem cell line building technology has been continuously enriched and improved,But for more than 20 years, the species that have been able to subculture the reproductive line and have established a specific line are still limited to a limited number of strains of mice, LIF, and the serum-containing culture system of the feeder layer, which cannot be used in all mouse embryos and other species.Research team led by Chinese scientist Dr Yingzilong in 2008 successfully established rat embryonic stem cell lines into more small cells using 2i small molecular culture system based on the theory of "embryonic stem cell self-renewal ground state"The establishment of embryonic stem cells from mouse strains and even other species has brought new theoretical and technical support.In this study, one of the hotspots of medical research, the animal model of senile diseases, early aging mice P1 type, or SAMP1 mice, was used as the research object.The purpose of this study was to obtain embryonic stem cells from isolated SAMP1 mouse blastocysts by using a new serum-free 2i culture system supplemented with small molecules, and to successfully establish the embryonic stem cell lines of SAMP1 mouse disease model.The establishment of SAMP1 mouse embryonic stem cell line is of great significance.The application of 2i small molecule culture system to the successful establishment of the universal cell culture system has proved that the study of embryonic stem cells in mice with this disease model is helpful to understand the genetic characteristics of premature senescence and to identify disease-related genes.According to the mechanism of premature aging, presbycusis and other related diseases, gene correction technology is carried out, which lays the foundation for the final treatment and solution of these diseases.In this experiment, SAMP1 mouse blastocysts were isolated and cultured, and the obtained embryonic stem cells were infected with the virus.1.The blastocysts of 3.5d SAMP1 mice could be naturally hatched in 2i culture condition and ICMS-like clones could be obtained by further culture.2.These embryonic stem cell like clones were identified as embryonic stem cells by AKP staining, immunofluorescence staining and identification criteria of Reverse Transcription PCR.3.SAMP1 embryonic stem cells infected with pCDNA-GFP lentivirus could be used to obtain SAMP1 mouse embryonic stem cells with green fluorescent protein gene.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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