不同代昆明小鼠胚胎成纤维细胞的生长特性研究
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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/m L、20μg/m L和30μg/m L)分别处理MEFs 1、2、3 h,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/m L丝裂霉素C处理2 h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/m L,20 μg/m L and 30 μg/m L) 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 to3 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/m L 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.
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/m L,20 μg/m L and 30 μg/m L) 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 to3 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/m L 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.
引文
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[2] Saba-El-Leil MK,Frémin C,Meloche S. Isolation of mouse embryonic stem cell lines in the study of ERK1/2 MAP Kinase signaling[J].Methods mol Biol,2017,1487:243-253. doi:10. 1186/s13287-015-0024-2.
[3]Pei H,Fu HY,Hirai H,et al. Generation of induced pluripotent stem cells from Chinese hamster embryonic fibroblasts[J]. Stem Cell Res,2017,21:132-136. doi:org/10. 1016/j. scr. 2017. 04. 010.
[4]Lalit PA,Rodriguez AM,Downs KM,et al. Generation of multipotent induced cardiac progenitor cells from mouse fibroblasts and potency testing in ex vivo mouse embryos[J]. Nat Protoc,2017,12(5):1029-1054.doi:10. 1038/nprot. 2017. 021.
[5] Xu J. Preparation,culture,and immortalization of mouse embryonic fibroblasts[J]. Curr Protoc Mol Biol,2005:Unit 28. 1. doi:org/10. 1002/0471142727. mb2801s70.
[6]张学红,柳立军,薛石龙,等.小鼠胚胎成纤维细胞制备方法对比[J].兰州大学学报,2014,40(2):10-15. doi:10. 13885/j. issn.1000-2812. 2014. 02. 003.
[7]张小梅,方廖琼,王智彪.小鼠成纤维细胞饲养层的制备及C57BL/6小鼠胚胎干细胞系的建立[J].吉林大学学报,2012,38(6):1081-1085. doi:10. 13481/j. 1671x. 2012. 06. 014.
[8]胡三强,王妍妍,马勇宾,等.小鼠胚胎成纤维细胞的分离培养及饲养层制备[J].中国组织工程研究,2014,18(45):7306-7311. doi:10. 3969/j. issn. 2095-4344. 2014. 45. 017.
[9]王胜洁,景瑾,杨万路,等. C57BL/6小鼠胚胎成纤维细胞的分离培养与纯化鉴定[J].黑龙江畜牧兽医,2015,2(1):1-4. doi:10.13881/j. cnki. hljxmsy. 2015. 0159.
[10]王志强,梁锐,陈明清,等.胰蛋白酶消化法和组织块法原代培养包皮成纤维细胞的比较[J].生命科学研究,2012,16(3):242-247.
[11]叶珏,张燕婉,时娜,等.小鼠胚胎成纤维细胞的原代培养及饲养层制备[J].标记免疫分析与临床,2013,20(1):46-48. doi:10. 11748/bjmy. 1006-1703. 2013. 01. 014.
[12]Li Y,Li L,Chen ZN,et al. Engineering-derived approaches for i PSC preparation,expansion,differentiation and applications[J]. Biofabrication,2017,9(3):032001. doi:10. 1088/1758-5090/aa7e9a.
[13]Tee YH,Shemesh T,Thiagarajan V,et al. Cellular chirality arising from the self-organization of the actin cytoskeleton[J]. Nat Cell Biol,2015,17(4):445-457. doi:org/10. 1016/j. ydbio. 2015. 10. 019.
[14]Peckham M. How myosin organization of the actin cytoskeleton contributes to the cancer phenotype[J]. Biochem Soc Trans,2016,44(4):1026-1034. doi:10. 1042/BST20160034.
[15]郝同扬,陈世豪,张光景,等.丝裂霉素C对ICR小鼠胚胎成纤维细胞饲养层制备的影响[J].上海畜牧兽医通讯,2013(4):10-12. doi:10. 3969/j. issn. 1000-7725. 2013. 04. 004.