摘要
目的建立小鼠胚胎成纤维细胞(MEF)滋养层,用于胚胎干细胞的培养。方法取12．5～14．5胎龄的胎鼠分离原代成纤维细胞,在37℃时,用0．25%胰蛋白酶(含0．02%EDTA)消化组织块5 min,培养3 d后传代。采用20mg/L丝裂霉素C处理胚胎成纤维细胞2～4 h,或γ-射线21 Gy照射胚胎成纤维细胞1 h后制备出的滋养层细胞,均能有效地抑制胚胎干细胞的分裂,且不影响其活力。结果胎鼠分离原代成纤维细胞经20 mg/L丝裂霉素C处理或γ-射线21 Gy照射后细胞仍保持分泌多种生长因子的能力,在12 d内既不增殖,也不死亡。结论这种方法制备的滋养细胞层适用于胚胎干细胞的培养。
Objective To establish mouse embryonic fibroblasts as the feeder layers for supporting embryonic stem cells in vitro. Methods The feeder layers of mouse primary embryonic fibroblasts was isolated from the mouse fetuse of 12.5-14.5 day gestation, and the fetal tissue was digested for 5 min with 0.25 % trpsin containing 0.02 % EDTA at 37 ℃. And then the third passage of mouse embryonic fibroblast was treated with mitomycine （MMC） for 2-4 h or 21 Gy-irradiation for 1 h. Results After treated by MMC （20 mg/L） or γ-irradiation （21 Gy）, mouse embryonic fibroblasts were only cultured within 12 days with the capability to secrete multiple growth factors, neither proliferation nor apoptosis. Conclusion Preparaing mouse embryonic fibroblasts as feeder layers by means of this measure can be used for culturing embryonic stem cells in vitro.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2006年第9期1121-1123,i0006,共4页
Chinese Journal of Experimental Surgery
基金
国家重点基础研究发展规划资助项目(2005CB522603)
国家自然科学基金(30230370
30400172)
关键词
胚胎成纤维细胞
胚胎干细胞
滋养层
Embryonic fibroblasts
Embryonic stem cell
Feeder layer
