背景:胚胎干细胞是从动物早期胚胎的内细胞团或原始生殖细胞分离出来的具有发育全能性的一种未分化的无限增殖细胞系。而鸡胚胎干细胞则是从X期鸡胚的胚盘分离而来。目的:优化鸡胚胎干细胞分离方法和离体培养体系。方法:采用滤纸纸环-发环的方法从X期鸡胚分离胚盘细胞,并采用STO细胞作为饲养层和大鼠肝细胞(brl)条件培养基(CM)+细胞因子作为离体培养体系对分离的胚盘细胞进行培养。结果与结论:滤纸纸环-发环法获得的完整胚盘率为75%~85%,克隆形成率约为50%。brl-CM+饲养层培养体系,鸡胚胎干细胞可传至7代,而brl-CM+饲养层+细胞因子培养体系,鸡胚胎干细胞可传至25代。分离到的鸡胚胎干细胞,经碱性磷酸酶染色、SSEA-1染色鉴定,表明鸡胚胎干细胞处于未分化状态。提示,实验不仅优化了鸡胚胎的分离方法,获得完整且杂质少的胚盘,而且进一步优化了鸡胚胎干细胞体外培养体系。BACKGROUND:Embryonic stem cells are undifferentiated permanent cell line derived from inner cell mass cells and primordial germ cells of animal's early embryos.Chicken embryonic stem cells are derived from the blastodermal of a X-stage embryo.OBJECTIVE:To optim the separation method and in vitro cultural system of chicken embryonic stem cells.METHODS:The X-stage chicken embryos were isolated by using a small square of ?lter paper with a hole punched in the center,and the blastodermal cells were isolated by using the hair loop.STO cells were used to make feeder layer;at the same time,BRL-CM and cytokine were also used for chicken embryonic stem cells in vitro cultural system.RESULTS AND CONCLUSION:The filter paper loop and the hair loop could obtain complete the blastoderm,and the successful percentage was 75%-85%.The colony formation rate was about 50%.After culture in the BRL-CM + feeder layer + cytokine culture system,the passage of CES cells is the seventh generation;BRL-CM + feeder layer + cytokines,cultured chicken embryonic stem cells could passage to the 25th generation.Isolated chicken embryonic stem cells were in an undifferentiated state detected by alkaline phosphatase staining and SSEA-1 staining.The findings indicate that this experiment not only optimized the isolation method of chicken embryonic stem cells to obtain complete and pure embryos,but also further improved the in vitro culture system of chicken embryonic stem cells.国家973项目(2009CB941600)资助;国家自然科学基金项目(31072101)资助---
