支持牛类胚胎干细胞发育的饲养层培养体系的建立

Establishment of Feeder Layer Culture System Supported Development of Bovine Embryonic Stem Cells

以小鼠胎儿和牛睾丸为材料 ,以含 1 5 % NBS、0 .1 mmol/ Lβ-巯基乙醇、0 .1 μmol/ L Na2 Se O3的DMEM溶液为培养液 ,分离获得了传 1 5代的小鼠胎儿成纤维细胞和 5代牛睾丸成纤维细胞 ,建立了小鼠和牛类 ES细胞培养体系。结果表明 :牛睾丸成纤维细胞和小鼠胎儿成纤维细胞均属附着生长型细胞 ,与小鼠胎儿成纤维细胞相比较 ,牛成纤维细胞直径和长度大 ,生长速度快 ,易于老化 ;1 2～ 1 6日龄的小鼠胎儿最适宜分离与克隆小鼠胎儿成纤维细胞 ;在 2 5℃条件下 ,以 0 .2 5 %胰蛋白酶 +0 .0 4% EDTA消化液作用胎儿小块组织分离原代小鼠胎儿成纤维细胞 ,消化液作用时间不应超过 2 0 min,以相同的消化液在 3 7℃条件下 ,离散贴壁成纤维细胞 ,作用时间以 2～ 3 min为宜 ;培养细胞密度与传代时间间隔有密切关系 ,若传代时间间隔为 3～ 4d,培养细胞浓度应为 3× 1 0 5个 / ml～ 5× 1 0 5个 / ml;在成纤维细胞分离与克隆过程中 ,培养基中添加0 .1μmol/ L Na2 Se O3+0 .1 mmol/ Lβ-巯基乙醇 +1 5 % NBS,有利于 MEF和

The culture system of feeder layer on Bovine embryonic stem cells were established in DMEM culture medium supplemented with 0.1 μmol/L Na 2SeO 3,15% NBS and 0.1 mmol/L β mercaptoethanol(Sigma).The results showed that 5 passage murine embryonic fribroblasts (MEF) and 5 passage new bovine testicular fibroblasts (NBTF) were isolated and cloned;Comparing with MEF, the length and width of NBTF were biger, inddition to, NBTF cell growed fast; Murine embryos from 12d to 16d were most suitable for isolation and clone of MEF; Treated with 0.25%trypsin+0.04% EDTA, single MEF cell and single NBTF cell were isolated and cloned by short term (from 2 min to 3 min); To supplement with 0.1 μmol/L Na 2SeO 3, 15% NBS and 0.1 mmol/L β mercaptoethanol is beneficial to reproduction of MEF and NBTF cells.