影响体外培养兔胚发育和兔类ES细胞分离的若干因素

本试验系统地比较了影响兔囊胚体外培养和免类ＥＳ细胞系分离的几个主要因素。结果表明，３种不同糖浓度（４．５，１．０，０．２ｇ／Ｌ）在４８ｈ内对胚胎贴壁均无显著影响。在高糖ＤＭＥＭ中，早期胚胎内细胞团（ｉｎｎｅｒｃｅｌｌｍａｓｓ，ＩＣＭ）增殖速度最快，分化也快；超低糖组，ＩＣＭ增殖缓慢，分化出现时间较晚；低糖组，ＩＣＭ既能保持较快的增殖速度，又能维持较长时间的未分化状。高糖组和低糖组的ＥＳ细胞样集落的传代能力相似，可达６～７代，超低糖组中不超过２代。胰岛素能促进ＩＣＭ的增殖，并可提高传代过程中ＥＳ细胞样克隆的形成率。在小鼠胚胎原代成纤维细胞（ＭＥＦ）和一种经白血病抑制因子转化了的小鼠成纤维细胞系（ＳＮＬ）上，ＩＣＭ都能维持较快的增殖速度，在家兔胚胎原代成纤维细胞（ＲＥＦ）中ＩＣＭ增殖较慢，无饲养细胞条件下，ＩＣＭ存活率低，生长状况不佳。传代能力，ＲＥＦ要比ＭＥＦ和ＳＮＬ低，且主要形成上皮样集落。作者根据以上实验结果认为，用低糖ＤＭＥＭ为基本培养基，再加入胰岛素，在ＭＥＦ或ＳＮＬ上培养切割胚胎和连续传代的技术路线是较为可行的。

Several Factors Affecting Development of Rabbit Embryosin Vitro and Establishment of Rabbit Embryonic Stem(ES)Cell-like Cell Lines

To make out optimum strategies for the isolation of rabbit ES cell lines,several factors related to establishment of rabbit ES cell lines have been studied compar-atively.Speed of proliferation and differentiation of inner cell masses (ICMs)was thefastest in DMEM with high glucose (4.5g/L)and was the slowest in DMEM with su-per-low glucose (0.2g/L). ICM cultured in low-glucose (1.0g/L)medium kept fastproliferation and long-time undifferentiated state,About 20 colonies could be observedwhen ICMs were passaged initially in the high- glucose or low- glucose medium, but pro-portion of ES- like cell colonies was very low in the high- glucose medium. No more than10 colonies were seen after initial passage in the super- low glucose medium.The abilityof ES-like cell colonies to survive from repeated passages was similar in the high-glucoseand low- glticose medium.The colonies passaged 6 to 7 times were obtained in high andlow-glucose media,but more than 2 passage colonies couid not be obtained in super-lowglucose medium.Insulin promoted proliferation of ICM cells and increased the numberof ES- like cell colonies during subcultures.ICMs living on MEF and SNL proliferatedwith similar speed, but faster than on REF.During the process of initial sub- Culture,new colonies on REF was fewer than on MEF and SNL,and most of them belonged toepithelial- like cells.According to our results,adoption of low-glucose as basic medium,addition of insulin and selection of MEF or SNL as feeder layer might be the optimumstrategy for isolation of rabbit ES cell lines.