The methods of evans and martin were changed slightly and used to isolate the mouse es cell in my experiment . in brief , the intact blastocysts were plated on sto feeder layer treated with mitomycin , and were cultured in the media supplymented with brl condition medium 联合evans和martin的方法，稍加改良来分离小鼠胚胎干细胞，把昆明白小鼠完整的囊胚直接种植在经丝裂霉素灭活的sto饲养层细胞上，在含有brl条件培养基的es细胞培养液中培养。
The results showed that the feeder layers prepared by icr and kunming mice had no significant effect on adherence of embryos and proliferation of icms . the trypsin , which affected the isolation of es cells remarkedly , should be used with concentration of 0 . 125 % trypsin + 0 . 02 % edta for kmsmin 从实验结果来看， icr小鼠和昆明小鼠两种来源的饲养层细胞对胚胎贴壁、 icm增殖无影响，消化液作用影响明显，以0 . 125 trypsin + 0 . 02 edta消化10 15min为宜。
Part 1 : the culture and identification of es - d3 cells and the study of the efficiency of eb formation from es cells when grown on mef feeder layer in es culture medium or cultured in es culture medium supplemented with lif 1000u / ml , es - d3 cells being used in our experiments formed normal clones , expressed akp and kept their normal karyotype over many passages . the in vitro and in vivo differentiation experiments showed that es - d3 cells could differentiate into variety of cell types derived from three primary germ layers 结果显示： eso3细胞在小鼠胚胎成纤维细胞上和或含白血病抑制因于亿f ）的es细胞培养液中形成典型的胚胎干细胞克隆，碱性磷酸酶染色结果为强阳性，具有正常二倍体核型以及具有在体内外分化为三个胚层来源的组织细胞的潜能，而且具有形成种系嵌合动物的能力。
( 7 ) in order to establish a feeeder - free system in porcine eg culture , we put pgcs on sto feeder layer , on sto extracellular matrix or on 0 . 1 % gelatin in brl - cm , and the results showed that when pgcs cultured on sto feeder , the brl - cm had a positive effect on keeping the undifferent state of eg colonies ( 7 )对非饲养层培养系统培养猪pgc进行了初步探索。实验结果证明，以sto为饲养层，培养液中加条件培养基对猪eg细胞保持不分化状态有积极作用，但培养板底铺有sto基质或明胶，即使用条件培养基也没有获得典型的eg集落。
After 4 days of growth , the intact inner cell mass ( icm ) were separated with 0 . 05 % trypsin - edta and replated on feeder layer in dmem containing 15 % serum , 0 . 1mmol / l nonessential amino acids , 0 . 1mol / l 2 - mercaptoethanol , 2mmol / l glutamine , 100 units / ml of streptomycin and 100 units / ml of penicillin . after 4 - 6 days 5 es cell colonies were selected and expanded in which alkaline alkaline phosphatase was detected ( 2 )小鼠囊胚或内细胞的培养和es细胞的分离培养了156个不同品系的小鼠囊胚，经过3 ? 4天培养后，将增殖出来内细胞团用机械法结合胰酶- edta处理，离散后培养于mef饲养层上， 4 6天后有5例出现了es细胞样集落。
( 5 ) when pgcs were cultured on different feeder layers , the better results were got if pgcs were cultured on sto and mef1 feeder layers , when pgcs were cultured on thawed sto feeders , pef , inactivated pef feeders , 0 . 1 % gelatin , we can not get typical eg colonies ( 5 )用不同饲养层培养pgc ，实验结果证明pgc在新鲜制作的sto和mef ~ 1饲养层上，可以得到传3代以上的eg集落； pgc在铺有解冻的sto饲养层细胞、 pef或丝裂霉素c处理过的pef 、涂有0 . 1明胶的4孔板上，一直没有得到典型的eg集落。
Medium which is serum - free or in the low concentration serum inhibit the proliferation of cells . a certain degree of increase serum concentration can promote the growth rate as well as prolong the life . the feeder layer cells were provided by mef , mue ( mouse uterus epithelium ) and rmc ( rat myocardial cells ) Mef和es细胞体外培养血清浓度以15 20为宜，试验中以mef 、 mue 、 rmc作饲养层，结果表明：以12 . 5dpc的mef为饲养层，无论是在小鼠es细胞的原代培养，还是在克隆传代过程中，效果都是最佳的。
We used the icr and kunming mice and got the embryos of 3 . 5dpc by means of superovulation , then cultured the embryos on the feeder layer derived from icr or kunming mice , dispersed the inner cell masses ( icms ) or es cell colony in the appropriate time . in this period , we analized the effects of feeder layer , trypsin and embryos isolated from uteri of different varietal mice on the es cell lines 以icr小鼠和昆明小鼠为实验对象，采用超数排卵的方法获得小鼠胚胎，培养在用丝裂霉素c处理过的icr小鼠和昆明小鼠mef细胞饲养层上，选取适当时间离散增殖的icm和类es细胞集落，分析了在小鼠es细胞建系过程中，饲养层细胞、消化液及不同品系来源的胚胎的影响。
( 3 ) isolation and culture of human primordial germ cells ( pgcs ) . human pgcs collected from gonadal ridges and mesenteries were grown on mouse feeder layers in the presence of human recombinant leukemia inhibitory factor ( lif ) , human recombinant basic fibroblast growth factor , and forskolin as described previously . initially , pgcs were visualized by alkaline phosphatase activity staining ( 3 )人类pgcs的分离和培养从4 10周龄药物流产胚胎的生殖嵴和肠系膜组织中分离原始生殖细胞( primordialgermcells ， pgcs ) ，培养在添加人重组白血病抑制因子( lif ) 、人重组碱性成纤维细胞生长因子( bfgf )和forskolin的小鼠饲养层细胞上。
The primary primordial germ cells is obtain from the human embryos after 4 - 8 weeks of pregnancy . after mechanical desection and enzymic digestion , the cells were cultured on mouse embryonic flbroblast or human embryonic flbroblast feeder layer inactivated by mitomycin . the medium contains several cytokines : lif ( leukemia inhibitory factor ) , bfgf and foskolin 从4 ? 8周的流产胎儿的原始生殖嵴部位分离到原始生殖细胞，经过机械和化学方法的解离后培养于事先用丝裂霉素处理过的小鼠胚胎成纤维细胞或人胚胎成纤维细胞饲养层上，培养基中添加了lif ， bfgef ， foskolin等细胞因子，此后大约每7天左右传代一次。
