| 摘要: |
| 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4～7 d,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12(18.8%),8(12.5%),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14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(25.0%,22.2%)无显著性差异（P>0.05）。(2) 7，8和9日龄胚胎随胚龄增加,胚胎贴壁率(5.9%,57.1%,87,9%)和ICM形成率(0.0%,28.6%,81.8%)升高，差异显著（P<0.01）。(3) 9日龄囊胚直径0.8～1.2 mm培养36～48 h贴壁,贴壁率100%，ICM形成率100%；直径>1.2 mm囊胚贴壁时间为48～72 h,贴壁率57.1%,ICM形成率57.1%,差异显著（P<0.05）。去除直径>1.2 mm囊胚部分滋养层细胞培养36～48 h，贴壁率91.7%,ICM形成率83.3%;(4) 添加外源LIF没有提高贴壁率(86.4%,90.9%;P>0.05)和ICM形成率(81.8%,81.8%;P>0.05)。 |
| 关键词: 胚胎干细胞 内细胞团 囊胚 分离 猪 |
| DOI： |
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| 基金项目:农业部生物技术项目(攻关)(85-11-02-1) |
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| Isolation of embryonic stem cells from inner cell mass of porcine blastocysts |
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| Abstract: |
| 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). |
| Key words: embryonic stem cells inner cell mass blastocysts isolation porcine |
