本文選題：胚胎干細胞　切入點：SAMP1小鼠　出處：《浙江理工大學(xué)》2011年碩士論文

【摘要】：自1981年小鼠胚胎干細胞成功建系以來,人們對胚胎干細胞的研究不斷深入:(1)胚胎干細胞已成為研究細胞分化、器官發(fā)生以及了解胚胎發(fā)育機制的有效手段;(2)對胚胎干細胞進行基因操作有助于確定特定基因在發(fā)育、生理和病理過程中的作用;(3)成功建系人類疾病動物模型的胚胎干細胞,結(jié)合近年來興起的基因打靶(基因敲除或基因敲入)等生物技術(shù),可以研究人類高血壓、早衰等多種自發(fā)性疾病的發(fā)生機制,并對其治療和預(yù)防提供科學(xué)依據(jù)。胚胎干細胞建系是進行所有這些研究的前提和基礎(chǔ)。雖然胚胎干細胞建系技術(shù)已經(jīng)獲得了不斷的豐富和完善,但是二十多年以來能夠進行生殖系傳代明確建系的種屬仍然僅限于小鼠有限的幾種品系,LIF加飼養(yǎng)層的有血清培養(yǎng)體系并不能通用于所有的小鼠及其他物種的胚胎干細胞建系工作。2008年華人科學(xué)家應(yīng)其龍博士領(lǐng)頭的研究小組基于“胚胎干細胞自我更新基態(tài)”理論利用2i小分子培養(yǎng)體系成功建立大鼠胚胎干細胞系為更多的小鼠品系甚至其它物種的胚胎干細胞建系工作帶來了全新的理論與技術(shù)支持。 本實驗以醫(yī)學(xué)研究熱點之一的老年性疾病的動物實驗?zāi)Ｐ驮缢バ∈驪1型即SAMP1小鼠作為研究對象,旨在通過應(yīng)用新型的添加小分子的無血清2i新型培養(yǎng)體系對分離的SAMP1小鼠囊胚的內(nèi)細胞團(ICM)進行培養(yǎng)而獲得胚胎干細胞,成功建立SAMP1小鼠疾病動物模型的胚胎干細胞系。建立該疾病模型SAMP1小鼠胚胎干細胞系具有重大意義(:1)目前國內(nèi)外從未有過早衰小鼠模型胚胎干細胞成功建系的報道,應(yīng)用2i小分子培養(yǎng)體系建系成功很好的驗證了這種培養(yǎng)體系的通用型;(2)對這種疾病模型小鼠胚胎干細胞的研究有助于人們清楚了解早衰的遺傳學(xué)特征,從而確定疾病相關(guān)基因,并實施基因打靶等基因矯正技術(shù),根據(jù)過早衰老、老年性耳聾等相關(guān)疾病的發(fā)生機理有的放矢,對癥下藥,為這類疾病最終的治療和攻克奠定基礎(chǔ)。 本實驗通過分離SAMP1小鼠囊胚內(nèi)細胞團(ICM)、對其進行培養(yǎng)并對獲得的胚胎干細胞進病毒感染,得出: 1. 3.5d SAMP1小鼠囊胚可以在2i培養(yǎng)條件下自然孵化出內(nèi)細胞團(ICM),進一步培養(yǎng)可獲得類胚胎干細胞樣克隆; 2.對這些類胚胎干細胞樣克隆進行AKP染色,Oct4,Sox2,SSEA-1基因表達免疫熒光染色以及Reverse Transcription PCR等胚胎干細胞的鑒定標準試驗,表明這些呈克隆生長的細胞即為胚胎干細胞; 3.用pCDNA-GFP慢病毒感染獲得的SAMP1胚胎干細胞,可以得到持久攜帶綠熒光蛋白基因的SAMP1小鼠胚胎干細胞,傳代次數(shù)30代以上仍可觀察到熒光。
[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.
【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】：2011
【分類號】：R329

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