猪胚胎干细胞培养、分离和传代

摘要:利用五指山小型猪近交系不同发育阶段的早期胚胎为材料，探索猪胚胎干细胞(embryonic stem cells,ES)培养、分离、传代的影响因素，以选择猪胚胎干细胞建系的适宜条件。收集五指山猪第5～10 d胚胎(配种当天记为0 d)，以 STO细胞[STO细胞是来自SIM小鼠(S)胚胎对硫代鸟嘌呤(thioguanine,T)和乌本苷(ouabain,O)有抗性的成纤维细胞系]为饲养层，分别采用两种培养液: 杜氏培养液(Dulbecco's modified eagle medium ，DMEM) + 10%胎牛血清（fetal bovine serum，FBS）+ 10 %新生牛血清（newborn bovine serum，NBS）+1 000 IU/mL白血病抑制因子（leukemia inhibitory factor，LIF）+ 30 ng/mL干细胞生长因子（stem cell growth factor , SCF）+20 ng/mL碱性成纤维细胞生长因子(basic fibroblast growth factor ，bFGF)或DMEM + 10% NBS + 10% FBS + 1 000 IU/mL LIF + 30 ng/mL SCF。比较发现，培养液中是否加入bFGF对胚胎干细胞的培养无显著影响；实验共收集胚胎124枚，其中D5～6胚胎18枚，胚胎贴壁率为68.8% ，内细胞团出现率为6.3% ，未能传代；收集D7孵化囊胚27枚，贴壁率为100% ，内细胞团出现率为38.9% ，传代1次失败；D9胚胎18枚，贴壁率为100%，传代后ES细胞生长较缓慢；收集D10胚胎52枚，胚胎贴壁率为100%，传代率为100%，传代后可出现ES细胞克隆点；实验过程中比较了胚径对干细胞培养的影响，发现胚径越大，培养效果越好，胚径大于100μm,分离ICM后出现干细胞集落成功率达100%，并得到了AP染色呈阳性的传4代的ES细胞集落。

Culture, Isolation and Passage of Porcine Embryonic Stem Cells

Abctract: Embryonic stem (ES) cells are pluripotential cells isolated from culture of preimplantation embryos in vitro. Experiments were undertaken to identify preimplantation embryonic stages and culture conditions under which pluripotential porcine embryo-derived cell lines could be isolated. Porcine embryos from D5,6,7,9,10 after the first day of estrus and insemination (day 0) were recovered by a surgical uterine flush technique with phosphate buffered saline (PBS) supplemented with 1% fetal bovine serum (FBS). A total of 124 porcine embryos were collected. Embryos were cultured at 38 ℃ on the feeder layers prepared from mitotically inactivated permanent mouse embryonic fibroblasts (STO) in the same medium[DMEM(Dulbecco's modified eagle medium) + 10 % NBS (newborn bovine serum)+ 10 %FBS+1 000 IU/mL LIF (leukemia inhibitory factor)+ 30 ng/mL SCF（stem cell growth factor）], the ICM(inner cell masses) growing rate of the embryos collected on day 5, 6, 7, 9, 10 was 0, 12.5%, 59.2%, 100%, 100% respectively; When embryos were cultured in two different mediums [Medium 1:DMEM + 10 % NBS + 10 % FBS + 1 000 IU/mL LIF + 30 ng/mL SCF; Medium 2: DMEM + 10 % NBS + 10 % FBS + 1 000 IU/mL LIF + 30 ng/mL SCF+ 20 ng/mL bFGF (basic fibroblast growth factor)], the result showed that bFGF was not essential in the culture and passage of ES cells; When the diameter of Day10 embryos was considered, we got that the larger embryos were better in ES cell culture, and the highest ES cells passages rate was gotten when the embryos have a diameter over 100 μm. In this experiment, we got ES-like colonies passed 4 times thatexpressed alkaline phosphatase activity.