由猪囊胚内细胞团分离胚胎干细胞的研究

Isolation of embryonic stem cells from inner cell mass of porcine blastocysts

以 PMEF为饲养层培养猪 7～ 9日龄囊胚分离 ES细胞 ,ICM初次传代时间为 4～ 7d,ES细胞传代时间为 4～ 5 d,ES细胞传至 1～ 5代的胚胎数分别为 1 2 (1 8.8% ) ,8(1 2 .5 % ) ,5 (7.8% ) ,3(4 .7% ) ,2 (3.1 % ) ,并进行体外分化和 AKP染色鉴定。对影响猪 ES细胞分离与克隆的因素进行比较 ,结果表明 :(1 ) 9和 1 4日龄小鼠胎儿 PMEF饲养层培养 9日龄猪囊胚 ,贴壁率 (83.3% ,88,9% ) ,ICM形成率 (75 .0 % ,77.8% )和 1代 ES细胞克隆率(2 5 .0 % ,2 2 .2 % )无显著性差异 (P>0 .0 5 )。 (2 ) 7,8和 9日龄胚胎随胚龄增加 ,胚胎贴壁率 (5 .9% ,5 7.1 % ,87,9% )和 ICM形成率 (0 .0 % ,2 8.6 % ,81 .8% )升高 ,差异显著 (P<0 .0 1 )。(3) 9日龄囊胚直径 0 .8～ 1 .2 m m培养36～ 4 8h贴壁 ,贴壁率 1 0 0 % ,ICM形成率 1 0 0 % ;直径 >1 .2 mm囊胚贴壁时间为 4 8～ 72 h,贴壁率 5 7.1 % ,ICM形成率 5 7.1 % ,差异显著 (P<0 .0 5 )。去除直径 >1 .2 m m囊胚部分滋养层细胞培养 36～ 4 8h,贴壁率 91 .7% ,ICM形成率 83.3% ;(4 )添加外源 L IF没有提高贴壁率 (86 .4 % ,90 .9% ;P>0 .0 5 )和 ICM形成率 (81 .8% ,81 .8% ;P>0 .0 5 )

Day 7 to day 9 porcine blastocysts were cultured on primary embryonic fibroblast (PMEF) feeder layer to isolate the embryonic stem (ES) cells.The optimum passage time of porcine inner cell mass (ICM) and ES cells was from day 4 to day 7 and day 4 to day 5,respectively.The number of embryos from which the ES cells were derived was 12(18.8%),8(12.5%),5(7.8%),3(4.7%) and 2(3.1%) from passage 1 to passage 5,respectively.The effect of the feeder layer,the embryonic age,diameter,and supplied LIF on the isolation of porcine ES cells was studied.The results were as followings:1) When day 9 porcine blastocysts were cultured with feeder layer of PMEF from day 9 and day 14 fetuses,there were no significant difference between the attachment rate (83.3%,88.9%),ICM formation rate (75.0%,77.8%) and passage 1 ES cells clone rate (25.0%,22.2%).2)The attachment rate and ICM formation rate from day 7 to day 9 blastocysts were5.9%,0.0%;57.1%,28.6%;87.9%,81.8% ( P <0.01).3) When the diameter of day 9 blastocysts was from 0.8 to 1.2 mm,the attachment time was 36-48 h,attachment rate 100.0% ,ICM formation rate 100.0%;diameter higher than 1.2 mm,48-72 h,57.1% and 57.1%,respectively ( P <0.01) .After cutting the blastocysts (day 9,diameter higher than 1.2 mm) and removing part of the trophoblasts cells,the attachment time was 36-48 h,attachment rate 91.7%,ICM formation rate 83.3% .4) The medium supplied with LIF did not increase the attachment rate (86.4%,90.9%; P >0.05) and ICM formation rate (81.8%,81.8%; P >0.05).