Optimization of separation methods and culture system of chicken embryonic stem cells in vitro

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---

圈养孟加拉虎脂肪间充质干细胞的分离培养和生物学特性

圈养孟加拉虎（Panthera tigris tigris）是保护该濒危物种的有效途径，但限制性的圈养环境导致圈养虎的健康问题日益凸显，尤其是肾炎，当前的治疗方法预后效果较差。为分离获取、鉴定并培养具有治疗孟加拉虎肾炎治疗潜力的孟加拉虎脂肪间充质干细胞，通过采集孟加拉虎的脂肪组织，利用Ⅰ型胶原酶消化法分离脂肪间充质干细胞并进行传代培养；采用CCK8法测定P3、P6和P9代细胞活性并绘制生长曲线；采用PCR法扩增间充质干细胞表面特殊标记物基因；通过成脂肪和成骨诱导分化试验分别测定脂肪间充质干细胞的成脂与成骨诱导分化能力。结果发现：孟加拉虎脂肪间充质干细胞为长梭形，折光性较好，生长情况良好；P3、P6和P9代细胞生长曲线呈典型的“S”型，符合间充质干细胞生长曲线规律；PCR扩增得到CD44、CD90、CD105和CD29特异性表面标记物基因；诱导分化试验表明，脂肪间充质干细胞具有成脂成骨诱导分化能力。综上，分离培养获得的细胞符合间充质干细胞特征，表明成功分离出孟加拉虎脂肪间充质干细胞。虎脂肪间充质干细胞的成功分离将作为一种潜在的细胞治疗手段，可能为虎肾炎的治疗提供新的选择，有望在维持虎的健康状态和延长寿命方面产生积极的临床效果