牦牛胎儿成纤维细胞的分离培养

Isolation and culturing in vitro of yak embryonic fibroblasts

采用30d胚龄左右的牦牛胚胎,用2种不同的消化液消化后,将所得的牦牛胎儿成纤维细胞(yak embryonic fibroblasts,YEF)培养于不同血清含量的RPMI1640培养液中,并接种于2种不同材质的培养瓶表面,对YEF的分离方法、培养所需胎牛血清(FBS)添加量和不同基质表面对YEF生长的影响进行了研究。结果表明,含150 mL/L FBS的RPMI1640培养基最适合YEF生长,可传代27次以上;经用RPMI1640和D-Hank’s液配制的2．5 g／L胰蛋白酶消化,所得的YEF的细胞活率差异不显著(P>0．05),其原代细胞在含100 mL／L胎牛血清的RPMI1640培养液中的贴壁率差异极显著(P<0．01);不同生长基质对YEF的形态没有显著影响。

The yak embryonic fibroblasts（YEFs） selected from 30days-old yak fetus were dispersed by different trypsin and cultured in RPMI1640 with different concentrations of fetal bovine serum（FBS）. The cells were attached to the glass or plastic and reached confluence. The YEFs was cultured successfully in RPMI1640 media with 150 mL/L of FBS and were passed more than 27 times in vitro. The viability of the primary YEFs dispersed by RPMI1640-trypsin and D-Hank＇s-trypsin solution were 93% and 89%, respectively. No significant difference existed between them. However, a significant difference was observed in the sticking efficiency of YEFs digested by D-Hank＇s-Trypsln （70. 50%） and by RPMI1640-Trypsin （84.7%）. No obvious effects were found on the shape of YEF when it was cultured in the different substrates.