摘要
目的分离培养昆明小鼠成纤维细胞,研究其体外生长特性并用于胚胎干细胞培养。方法消化法分离培养获得昆明小鼠胚胎成纤维细胞,利用MTT法绘制各代细胞生长曲线,免疫荧光对各代细胞进行细胞骨架分析;用不同浓度丝裂霉素C(10μg/mL、20μg/mL和30μg/mL)分别处理MEFs1、2、3h,MTT法筛选饲养层制备的最佳条件,制备饲养层用于小鼠胚胎干细胞的培养。结果分离的小鼠胚胎成纤维细胞3~5代细胞增殖能力较好;1~3代细胞微管微丝排列整齐,5代以后的细胞微管微丝排列紊乱;10μg/mL丝裂霉素C处理2h有利于小鼠胚胎干细胞的培养。结论体外分离培养的3~5代小鼠胚胎成纤维细胞可用于小鼠胚胎干细胞的培养。
Objective Isolation and culture of Kunming mouse embryonic fibroblasts (MEFs) for growth feature in vitro are used to culture the embryonic stem cell.Methods Kunming MEFs were isolated and cultured by digestion.MTT assay was used to plot the growth curve of each generation of cells.The cytoskeleton of each generation of cells was analyzed by immunofluorescence.The MEFs were dealt with different concentration of Mitomycin C(10 μg/mL,20 μg/mL and 30 μg/mL) for 1 hour,2 hours and 3 hours.The optimum condition of feeder layer preparation was screened by MTT assay.The feeder layer was prepared for the culture of mouse embryonic stem cells.Results The proliferation ability of generation 3 to 5 of MEFs was good.The cytoskeleton immunofluorescence staining showed that the generation 1 to 3 of cell microtubules and microfilaments arranged in neat rows,the generation 5 to 7 of cells microtubules and microfilaments were disorganized.The feeder cells dealt with 10 μg/mL Mitomycin C for 2 hours was better for the culture of mouse embryonic stem cell.Conclusion The generation 3 to 5 of MEFs can be used for the culture of mouse embryonic stem cells.
作者
黄悦
严会文
王燕燕
苏敏
HUANG Yue;YAN Hui-wen;WANG Yan-yan;SU Min(Department of Anatomy,Guiyang Nursing Vocational College,Guiyang Guizhou 550081,China;Department of Histology and Embryology,Guizhou Medical University,Guiyang Guizhou 550081,China)
出处
《局解手术学杂志》
2019年第7期520-524,共5页
Journal of Regional Anatomy and Operative Surgery
基金
贵州省科技厅基金[黔科合J字(2015)2011号]
贵州省优秀科技教育人才省长专项资金项目[黔省专合字(2012)41号]
