鸡胚胎干细胞分离培养和单细胞克隆的制备

Isolation and Cultivation of Chicken Embryonic Stem Cells and Production of Single-cell Clone

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、5.5×10-5mmol/Lβ-巯基乙醇、10μL/mL非必需氨基酸、以及含1000U/mLLIF、10ng/mLbFGF和5ng/mLSCF的高糖DMEM对细胞进行培养和传代。利用鸡第Ⅹ期的胚胎干细胞,采用口吸管法、细胞稀释法和克隆环法3种方法,制备单细胞克隆,采用细胞化学法和免疫荧光法检测细胞表面标志物。结果显示,可获得传至5～6代的鸡胚胎干细胞。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。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。口吸管法、细胞稀释法和克隆环法单细胞克隆形成率分别为0、4.2%和1.0%。经AKP活性和SSEA-1免疫荧光鉴定均呈阳性,扩增出的克隆能稳定增殖不分化。结果表明,细胞稀释法操作简单易行,试验时间短,对细胞伤害小,为鸡ES单细胞克隆进一步建系提供了可行的方法。

The isolated blastoderm cells from the stage X embryos of chicken were cultured on feeder layer of chicken embryonic fibroblasts and DMEM supplemented with 10% fetal bovine serum,2% chicken serum,2 mmol/L L- glutamine,5.5×10^-5 β-mercaptoethanol,l mmol/L sodium pyruvatel,with 1 000 U/mL LIF,IO ng/mL bFGF and 5 ng/mL SCF. Mouth sucker method, clone link method and cell limiting dilution method were compared in order to prepare single-cell clone from stage X ES ceils of chicken. Special markers of single-cell clone were detected by cytochemistry and histoimmunochemistry. The results showed that ES were subcultured to 5^th to 6^th generation. The cultured ES cells maintained characters of stem cells by AKP and SSEA-1 differentiation experiments. The different isolation methods and different digesting time were compared,the result showed spoon method could easily obtain blastoderm and the cells digested 5 to 8 rain grew better. Rates of single-cell clone of mouth sucker method,cell limiting dilution method and clone link method were 0,4.2% and 1.0% respectively. Single-cell clone was positive to AKP and SSEA-I. The results indicated that cell limiting dilution method was the best method of preparation for single-cell clone among three methods.