猪类胚胎干细胞建系过程中对培养液的优化研究

发布时间：2018-06-25 00:48

本文选题：猪 + 猪类胚胎干细胞　；参考：《东北农业大学》2015年硕士论文

【摘要】：胚胎干细胞是研究基因功能、建立疾病模型和促进再生医学领域发展的一种重要工具。猪由于生理结构与人类相似,长期以来被人们作为疾病模型广泛的应用于临床研究中,因此猪胚胎干细胞的建立对于生物医学领域有着重大意义。不仅如此,猪作为农业经济动物,其胚胎干细胞的建立可以在基因工程水平上来帮助改善猪的健康水平和生长性状提高经济效益。但在猪胚胎干细胞的研究中,人们始终没有获得一株真正的胚胎干细胞系,同时猪胚胎干细胞所需要的培养条件也不能确定,在2014年我们实验组获得了一株猪类胚胎干细胞系,512506具有一定的干细胞特点,其培养液(MXV培养液),经研究是一组比较适合猪类胚胎干细胞生长的干细胞培养液。但是由于其成分过于复杂,操作性差,并不利于猪胚胎干细胞的进一步研究。在猪胚胎干细胞培养条件的探索中,人们依据大鼠胚胎干细胞的成功,2i体系也得到了广泛的应用。但却存在着相反的两种作用效果。本实验针对MXV培养液的缺点,我们对其进行了优化,找出MXV培养液中必需成分。并探索了2i对猪胚胎干细胞建系的影响。首先是针对MXV培养液的五种主要成分bFGF,hLif,BSA,N2B27,KOSR,进行逐一简化,分别通过半量降低这五种成分的培养液和全量降低这五种成分的培养液分别培养512506细胞系最终可以去掉MXV培养液中的不重要成分。得到成分简化的MXV培养液,随后,我们对MXV培养液中基础培养液进行优化,X中的基础培养液为三种基础培养液Neurobasal,KO-DMEM,和DMEM/F1的混合物。我们用单一的培养液成分来替代这个混合体系。最后可以获得简化的MXV培养液。为了进一步确定简化的MXV培养液是否可以完全替代MXV培养液,本实验探索了简化MXV培养液对猪类胚胎干细胞建系效率的影响。同时针对CHIR99021,PD0325901简称2i对于有猪多能性细胞研究中相对立的研究结果,我们探究了2i对猪胚胎干细胞建系效率的影响。经过上述内容研究的结果显示:KOSR可以从MXV培养液中去除获得MXV-K培养液,不添加KOSR的优化MXV培养液可以使512506的细胞状态更好,细胞排列紧密,脂滴消失,形态更接近人胚胎干细胞系。其他三组主要成分虽然没有bFGF对MXV培养液那么大的影响,但去除后,512506细胞系状态仍然会变差。所以在五种成分中我们只能将KOSR去除.而针对基础培养液的简化,最终可获得两组单一的基础培养液KO-DMEM和DMEM/F12的优化MXV培养液,可以完全代替MXV培养液培养512506细胞系,获得的512506P5K和512506P5D在形态上比512506细胞系更接近人胚胎干细胞系,多能性状态与512506细胞系相似。在建系效率的研究中,实验结果显示发育至6d的体外受精胚胎在KO-DMEM培养液中贴壁率和原代克隆形成率显著高于DMEM/F12培养液,添加2i后两种培养液中的胚胎贴壁率和原代克隆形成率均下降。在KO-DMEM培养液中可获得稳定传代的细胞系,细胞形态与512506细胞系不同,无脂滴,细胞排列紧密,形态更类似与人胚胎干细胞系。细胞系呈碱性磷酸酶阳性,核型正常,表达Oct4、Sox2和Nanog,不表达Cdx2。细胞系可成功进行转基因操作。结果表明以KO-DMEM为基础培养液的优化MXV培养液体系可以获得猪类胚胎干细胞,2i不利于胚胎贴壁和原代克隆形成。该研究为猪胚胎干细胞建系培养体系选择与推断猪胚胎干细胞多能性细胞信号调控通路提供了重要的实验依据。
[Abstract]:Embryonic stem cells (SSCs) are an important tool to study gene function, establish disease models and promote the development of regenerative medicine. Pigs have been widely used as disease models in clinical research for a long time because of their similar physiological structure. Therefore, the establishment of pig embryonic stem cells is of great significance in the field of biomedicine. In this case, as an agricultural economy animal, the establishment of embryonic stem cells can help improve the health level and growth traits of pigs at the level of genetic engineering. However, in the study of pig embryonic stem cells, people have never obtained a real embryonic stem cell line, and the need for the cultivation of pig embryonic stem cells. The conditions were also undetermined. In 2014, a pig like embryonic stem cell line was obtained in our experimental group. 512506 had a certain characteristic of stem cells. The culture solution (MXV Culture) was a group of stem cell culture medium suitable for the growth of pig embryonic stem cells. But because of its complexity and poor operability, it was not beneficial to pig embryos. Further research on stem cells. In the exploration of the culture conditions of the pig embryonic stem cells, the 2I system has also been widely used on the basis of the success of the rat embryonic stem cells. But there are two kinds of effects. In this experiment, we have optimized the MXV culture solution to find out the essential components in the MXV culture solution. The effect of 2I on the construction of pig embryonic stem cell line was explored. First, the five main components of MXV culture solution, bFGF, hLif, BSA, N2B27, KOSR, were simplified one by one. The culture solution of the five components was reduced by half quantity and the 512506 cell lines of the five components were cultured respectively to remove the MXV culture solution. The important ingredient. We obtained the simplified MXV culture solution. Then, we optimized the basic culture medium in the MXV culture. The basic medium in X was the mixture of three basic cultures, Neurobasal, KO-DMEM, and DMEM/F1. We used a single culture medium to replace the mixture. Finally, we can obtain a simplified MXV culture. It is further determined whether the simplified MXV culture solution can completely replace the MXV culture solution. This experiment explored the effect of the simplified MXV culture on the establishment of the pig embryonic stem cell line construction efficiency. At the same time, we explored the establishment of 2I on the construction of pig embryonic stem cells according to the relative research results of CHIR99021 and PD0325901 for abbreviation of 2I in the study of porcine pluripotent cells. The results of this study showed that KOSR could remove MXV-K culture from MXV culture, and the optimized MXV culture solution without KOSR could make the 512506 cell state better, the cells were arranged closely, the lipid droplets disappeared, and the form was closer to the human embryonic stem cell line. The other three main components were not bFGF to MX. The effect of V culture is so great, but after removal, the state of the 512506 cell line will still be worse. So we can only remove the KOSR in the five components. For the simplification of the basic culture solution, we can finally obtain two sets of single basic culture medium KO-DMEM and DMEM/F12 optimized MXV culture, which can completely replace the MXV culture medium to cultivate 512506 cell lines. The obtained 512506P5K and 512506P5D were much closer to the human embryonic stem cell line than the 512506 cell line, and the pluripotent state was similar to that of the 512506 cell line. In the study of the building efficiency, the experimental results showed that the adherence rate and the primary clone formation rate of the in vitro fertilized embryos developed to 6D were significantly higher than that of the DMEM/F12 culture. After adding 2I, the rate of adherence to the embryo and the formation rate of primary clones in the two cultures decreased. In the KO-DMEM culture, a stable cell line was obtained. The cell morphology was different from that of the 512506 cell line. The cell line was closely related to the human embryonic stem cell line. The cell line was similar to the human embryonic stem cell line. The cell lines were alkaline phosphatase, the karyotype was normal, the expression of Oct4, S Ox2 and Nanog did not express the Cdx2. cell line successfully. The results showed that the optimized MXV culture liquid system with KO-DMEM as the base medium could obtain the pig embryonic stem cells, and 2I was not conducive to the formation of the embryo and the primary clones. The regulation of sex cell signaling provides an important experimental basis.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

【共引文献】