猪孤雌胚胎干细胞建系的研究

以猪孤雌激活囊胚为材料，囊胚透明带消化后采用全胚培养，培养液中添加不同培养成分或因子（如FGF2，LIF，2i等），以及选择不同的初始培养液体积来筛选猪胚胎干细胞（embryonic stem cell，ES细胞）建系的优化培养体系。囊胚内细胞团形成的细胞集落采用胰酶消化传代。结果显示：透明带消化后，囊胚贴壁率显著升高（19．4％VS．8．8％）（P〈0．05）；初始培养液体积比平常培养液体积（0．30mL／孔，24孔培养板）减半条件下，能显著提高其贴壁率（91．7％VS 20．0％）（P〈0．01），而且获得了可传至7代的类ES细胞系2株，碱性磷酸酶染色成阳性；当用2i因子（CHIR99021和PD03025901）去替代培养液中的FGF2，囊胚贴壁率（29．400VS53．3％）和原代集落形成率（20．0％VS 87．5％）反而显著下降（P〈0．01）。这表明培养液添加了FGF2和LIF（不舍2i因子），用24孔板培养，最初培养体积为0．15mL，透明带消化的培养体系比较适合猪孤雌激活胚的ES细胞建系。

Establishment of embryonic stem cell line derived from pig parthenogenetic blastocysts

This study was conducted to observe the effect of zona pellucida （ZP） digestion,different culture medium and their primary volume on the efficiencies of ES cell derivation from pig parthenogenetic blastocysts. Firstly,day 7 parthenogenetic blastocysts were digested with pronase to remove ZP and cultured on mouse embryonic fibroblast （MEF） feeder layer for inner cell mass （ICM） formation. Then the ES cell colonies from the ICM were disassociated by trypsin for passaging and subsequent propagation. The results showed that when the ZP was removed from the parthenogenetic blastocyst, the attachment rate （19.4%） was significantly improved （P〈0.05） compared with that （8.8%） with ZP. Secondly,when the primary culture medium was reduced to 0. 15 mL per well （24-well culture dish） ,the attachment rate was signifcantly （P〈0.01） higher （91.7%） than that （20.0%） of the embryos cultured in 0.30 mL of culture medium per well,and passage 7 ES cell eolony was obtained with positive alkaline phosphatase staining. Thirdly,when the FGF2 was replaced with 2i factors （CHIR99021and PD03025901） in the culture medi- um,the attachment rate （29.4% vs 53.3%） and ICM formation rate （20.0% vs 87.5%） from the parthenogenetic blastocysts were significantly lower （P〈0.01）. The data demonstrate that ES cell colonies disassociated by trypsin combined with the culture media containing FGF2 and LIF was suitable for the establishment of ES cell line from pig day 7 parthenogenetic blastocysts.