昆明小鼠胚胎干细胞培养体系的优化-临床兽医学专业论文.docxVIP

i 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(P0.01)；bFGF 、EGF 、肝素钠对小鼠胚胎干细胞 有一定的促进作用 (P0.05) ； SU5402 对小鼠胚胎干细胞 没有明显的促进作用 (P0.05)；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 ii ii Optimization of the culture system of Kunming mouse ESC Abstract This study investigated the effects of differentcultue systems, basal culture medium, cytokines, drugs, oxygen partial pressure and temperature on 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 research compared the effects of 2 kinds of culture systems (KSR and N2B27) on KM- ESC. The results showed that: ( i) in KSR culture system, ESC grew adherently and slowly and lots of cells on the fringe of colon ies 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 was smooth. No cell was 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 Stres s (5% O2, 20% O2) and temperature (37℃、 39℃)on culture of KM - ESC. The results s