鸡胚胎干细胞的分离和培养

Isolation and Cultivation of Chicken Embryonic Stem Cells

实验从鸡第X期胚胎中分离胚盘,以鸡成纤维细胞为饲养层,用添加了10%的胎牛血清、2%的鸡血清、2mmol/LL-谷氨酰胺、1mmol/L丙酮酸钠、5.5×10-5mol/Lβ-巯基乙醇、10μL/mL非必需氨基酸以及含1000IU/mL白血病抑制因子(LIF)、10ng/mL碱性成纤维生长因子(bFGF)和5ng/mL干细胞生长因子(SCF)的高糖DMEM对细胞进行培养和传代,可以获得传至5～6代的鸡胚胎干细胞(ES)。通过对传代培养后的鸡ES细胞进行AKP染色鉴定和SSEA-1的鉴定,证实细胞未发生分化,具有胚胎干细胞的特征。同时通过不同分离胚盘方法和不同消化时间的比较得出药勺法提取胚盘简单易行,原代消化5～8min的ES细胞适合于传代培养。

The isolated blastoderm cells from the stage X embryos of chicken were cultured on feeder layer of chicken embryonic fibroblasts and in DMEM supplemented with 10% fetal bovine serum, 2% chicken serum, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvatel, 5.5×10^-5 mol/L β-mercaptoethanol, with leukemia inhibitory factor （LIF, 1 000 IU/mL）, basic fibroblast growth factor （bFGF, 10 ng/mL） and stem cell factor （SCF, 5 ng/mL）, then the ceils were passaged and the sixth generation ES cells were obtained. The alkaline phosphatase （AKP） and SSEA-1 differentiation experiments were carried out, the results showed that the cultured ES cells maintain characters of stem cells. The effective of different isolation methods and different digesting time were compared, the result showed that using spoon can obtain blastoderm easily and the cells digested 5 to 8 min grow better.