昆明小鼠胚胎干细胞培养体系的优化.pdf

i昆明小鼠胚胎干细胞培养体系的优化摘要本试验旨在研究不同的培养方法、基础培养基、细胞因子、肝素钠、温度、氧分压等因素对昆明小鼠胚胎干细胞建系的影响,筛选了昆明小鼠胚胎干细胞的最佳培养条件,并以此为基础建立了携带绿色荧光蛋白(GFP)基因的小鼠胚胎干细胞系。试验利用两种不同的培养体系(KSR和N2B27)建立昆明小鼠胚胎干细胞。结果在KSR的培养体系中小鼠胚胎干细胞集落贴壁状态生长,生长较缓慢,边缘细胞分化脱落,最高传代为6代;在N2B27培养体系中细胞集落呈悬浮状态生长,生长较快,边缘光滑整齐,连续传7代未见分化。采用N2B27基本培养体系。研究不同基础培养液(高糖DMEM、knockout DMEM、DMEM/F12、TCM-199)、细胞因子(LIF、bFGF、EGF)、两种药物(SU5402、肝素钠)、温度和气相环境等因素对昆明小鼠胚胎干细胞的影响。结果表明,基础培养基knockout DMEM和DMEM/F12优于高糖DMEM和TCM-199;LIF对小鼠胚胎干细胞生长具有明显的促进作用(P<);bFGF、EGF、肝素钠对小鼠胚胎干细胞有一定的促进作用(P<);SU5402对小鼠胚胎干细胞没有明显的促进作用(P>);温度39℃比37℃更有利于小鼠胚胎干细胞的快速增殖,氧分压20% O2较氧分压5% O2有利于小鼠胚胎干细胞的建系。以N2B27系统为条件,采用卵周隙注射携带GFP基因的慢病毒载体法感染早期小鼠胚胎,再将转染小鼠胚胎接种在N2B27胚胎干细胞培养体系,经过培养获得携带GFP转基因的昆明小鼠胚胎干细胞。关键词:昆明小鼠;胚胎干细胞;培养基;细胞因子;温度;氧分压iiOptimization of the culturesystem ofKunming mouse ESCAbstractThis study investigated the effects of differentcultue systems, basal culture medium, cytokines, drugs, oxygen partial pressureand temperatureon establishment of Kunming mouse embryonic stem cell (KM-ESC). The optimum culture condition that is appropriate for growing of KM-ESC was selected. The KM-ESC carrying GFP gene was established subsequently. This pared the effects of 2 kinds of culture systems (KSRandN2B27) on KM-ESC. The results showed that:(i) in KSR culture system, ESC grew adherently and slowlyand lots of cellson the fringe of colonies were differentiated or apoptosis fell off. ESC could be passaged to 6 generation. (ii) In N2B27 culture system, ESC grew suspendedly and fastly. Edge of colony wassmooth. No cellwas differentiated when ESC was passaged to 7 generation. On the base of N2B27 culture system, this study reasearched the effects of 4 basal culture medias (DMEM-high glucose,knockout DMEM,DMEM/F12,TCM-199), 3 cytokines (LIF, bFGF, EGF), 2 drugs (SU5402, heparin sodium),Oxygen Stress (5% O2, 20% O2) and temperature (37℃、39℃)on culture of KM-ESC. The results showed that: knockout DMEM and DMEM/F12 were more appropriate for the growing of ESC than DMEM with high glucose and TCM-199. (ii) LI