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小鼠胚胎干细胞体外建系及电转染K14-EGFP基因的实验研究

发布时间:2018-01-16 08:12

  本文关键词:小鼠胚胎干细胞体外建系及电转染K14-EGFP基因的实验研究 出处:《福建医科大学》2008年硕士论文 论文类型:学位论文


  更多相关文章: 胚胎干细胞 皮肤细胞 诱导分化 电转染 角蛋白14 实验


【摘要】: 背景:目前,皮肤缺损是临床上经常遇到的问题,应用组织工程方法构建人工皮肤并应用于临床是研究的热点。在组织工程化皮肤的研究中,需要能快速大量扩增、长期体外传代培养、细胞功能旺盛的种子细胞。由于胚胎干细胞(Embryonic stem cells,ESC)有很高的再生及多向分化的潜能,以胚胎干细胞诱导分化而来的皮肤细胞为种子细胞构建组织工程化皮肤是一种很好的研究方法,而目前,国内外学者对胚胎干细胞诱导分化为皮肤细胞的深入研究并不多。 目的:本实验旨在研究和建立带有角蛋白10-增强型绿色荧光蛋白(Keratin14- Enhanced Green Fluorescence Protein, K14-EGFP)基因的小鼠胚胎干细胞系并进行鉴定,为组织工程化皮肤的基础研究及临床应用打下坚实的基础。 方法:首先体外建系129/S1小鼠的胚胎干细胞,并对其进行形态学、碱性磷酸酶染色(Alkaline Phosphatase,AKP)、阶段特异性胚胎表面抗原-1(Stage-specific embryonic surface antigen 1,SSEA-1)免疫组化染色、体内及体外分化实验等的鉴定分析,证明其胚胎干细胞的特性,再对建系成功的胚胎干细胞进行K14-EGFP基因电转染,并对转染成功的胚胎干细胞进行鉴定包括体内外向分化能力鉴定。 结果: 一.成功建立两株129/S1小鼠的胚胎干细胞系,并进行了形态学、碱性磷酸酶染色、阶段特异性表面抗原-1、体内及体外分化实验等的指标鉴定。 二.对建系成功的胚胎干细胞进行K14-EGFP(抗G418)电转染,经G418筛选的阳性克隆进行体外扩增,获得大量带有K14-EGFP基因片段的小鼠胚胎干细胞。 结论: 一.利用生长3.5天的小鼠囊胚分离培养胚胎干细胞,并且在建系培养过程中需应用MEF制备的饲养层和白血病抑制因子来促进胚胎干细胞的增殖和抑制其分化; 二.胚胎干细胞的电转染效率很低,但是转染成功的细胞经体外扩增可获得足够数量的转染阳性细胞; 三.转染K14-EGFP片段基因的胚胎干细胞经诱导有皮肤细胞表面标志物角蛋白14表达时就会有绿色荧光蛋白的表达。
[Abstract]:Background: at present, skin defect is a common problem in clinic. The application of tissue engineering to artificial skin is a hot topic in the research of tissue engineered skin. It needs seed cells that can be rapidly expanded, subcultured in vitro for a long period of time, and have high cell function. Because of embryonic stem cells, Embryonic stem cells. ESCC has a high potential for regeneration and multi-differentiation. It is a good method to construct tissue-engineered skin with skin cells derived from embryonic stem cells as seed cells. There are few researches on the induction and differentiation of embryonic stem cells into skin cells at home and abroad. Objective: to study and establish a new type of green fluorescent protein (GFP) with keratin 10 (K10)-enhanced green fluorescent protein (GFP). Keratin14-Enhanced Green Fluorescence Protein. The mouse embryonic stem cell lines of K14-EGFP gene were identified, which laid a solid foundation for the basic research and clinical application of tissue engineering skin. Methods: the embryonic stem cells of 129 / S1 mice were first established in vitro, and were morphologically stained with alkaline phosphatase (Alkaline Phosphatase). Stage-specific embryonic surface antigen 1. SSEA-1) immunohistochemical staining, in vivo and in vitro differentiation experiments, to prove the characteristics of its embryonic stem cells, and then successfully constructed embryonic stem cells for K14-EGFP gene electrotransfection. The successful transfection of embryonic stem cells was identified, including the ability of differentiation in vivo. Results: 1. Two embryonic stem cell lines of 129 / S1 mouse were successfully established and identified by morphology, alkaline phosphatase staining, phase specific surface antigen -1 and differentiation test in vitro and in vivo. 2. The successful embryonic stem cells were transfected with K14-EGFP (anti-G418), and the positive clones screened by G418 were amplified in vitro. A large number of mouse embryonic stem cells with K14-EGFP gene fragment were obtained. Conclusion: 1. Embryonic stem cells were isolated and cultured from mouse blastocysts for 3.5 days. The feeder layer and leukemia inhibitor prepared by MEF should be used to promote the proliferation and inhibit the differentiation of embryonic stem cells. 2. The efficiency of electrotransfection of embryonic stem cells is very low, but enough positive cells can be obtained by amplification of transfected cells in vitro. 3. The expression of green fluorescent protein was found in the embryonic stem cells transfected with K14-EGFP gene when it was induced to express keratin 14 on the skin cell surface.
【学位授予单位】:福建医科大学
【学位级别】:硕士
【学位授予年份】:2008
【分类号】:R329.21

【引证文献】

相关博士学位论文 前1条

1 梁洋;蜘蛛拖丝蛋白基因Spidroin1嵌合体小鼠的研究[D];东北林业大学;2011年

相关硕士学位论文 前1条

1 吕一帆;胚胎干细胞在内膜损伤小鼠宫腔内移植的实验研究[D];福建医科大学;2010年



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