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建立绿色荧光蛋白标记的小鼠胚胎干细胞系及向心肌样细胞的分化(英文) 被引量:1

Establishment of Murine Embryonic Stem Cell Line Carrying Enhanced Green Fluorescence Protein and its Differentiation into Cardiomyocyte-like Cells in vitro
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摘要 带有GFP基因的ESD3细胞系是一个良好的可以用于研究体内和体外细胞分化和组织产生的模型。用磷酸钙共沉淀法将质粒pEGFP N2导入小鼠胚胎干细胞D3细胞系中 ,在荧光显微镜下以 4 88nm激发光检查阳性克隆 ,并进行初步扩增。经G4 18筛选后 ,机械挑取EGFP强阳性表达的克隆 ,并在丝裂霉素C处理的小鼠胚胎成纤维细胞的饲养层上 ,在无选择性压力的条件下 ,进一步扩大培养 ,获得纯化的转染细胞系。 2 0代以后 ,转染细胞仍然表达绿色荧光蛋白。PCR检测表明 8代和 18代转染细胞均携带有GFP标志基因。对稳定表达EGFP的干细胞系进行碱性磷酸酶染色、拟胚体和畸胎瘤形成的检测 ,证明这些细胞具有干细胞的特征。经拟胚体 ,可进一步分化成具有搏动能力的心肌细胞 ,分化百分率为 30 %~ 4 0 % ,较未转染细胞 6 0 %~ 70 %的分化率低 ,造成低分化率机制还不清楚。这些细胞在激光共聚焦显微镜下呈绿色荧光 ,免疫组化染色显示具心肌细胞特异的cTnT分子标志。该EGFP标记的干细胞系带有可进行原位、实时检测的绿色荧光 。 The availability of EGFP ES cell D3 lines provided a tractable model to study cell differentiation and tissue generation in vivo and in vitro. Plasmid pEGFP N2 was introduced into the murine embryonic stem cell D3 by standard calcium phosphate precipitation. Transfected clones were screened out under the fluorescence microscope at the 488 nm emission light in the presence of G418. Strong fluorescent EGFP clones were singly picked out and further proliferated on a feeder layer of mitomycin-C treated mouse embryonic fibroblasts. One line of EGFP ES D3 cells subcultured twenty passages and still carried the EGFP DNA without the selecting pressure. It indicated that the gene might integrate into the ES genome or still dissociated in the cytoplasm. PCR analysis for EGFP DNA showed that undifferentiated EGFP ES cells at passage 8 and 18 carried the EGFP gene. Alkaline phosphatase staining, embryoid body and teratoma formation were performed to analyze the differentiation status and potential of the EGFP ES D3 cells. The cells derived from embryoid body were able to differentiate into beating cardiomyocytes with green fluorescence clearly observable under the confocal laser scanning microscopy. 30%~40% of cells from embryoid bodies were capable to differentiate into cardiomyocyte-like cells, and it appeared lower than the non-transfected ES D3 cells, which could be 60%~70% under the same conditions. The mechanism was currently unknown. Immunocytochemistry staining indicated that the contracting cells were cardiomyocytes based on the presence of cardiac specific molecular marker cTnT. Results showed that the stable EGFP positive ES cell line retained the typical characteristics of ES cells and possessed the pluripotential to differentiate into beating myocytes in vitro. The EGFP transfected cells stably yielding bright green fluorescence in real time and in situ rendered it was a powerful tool in cell transplantation and tissue engineering.
出处 《生物工程学报》 CAS CSCD 北大核心 2004年第6期890-895,共6页 Chinese Journal of Biotechnology
基金 浙江省重大项目 (No .J 2 0 0 2 0 5 79 3 0 116) 湖州中心医院合作项目 (No .H2 0 0 10 984 3 2 5 3 6)~~
关键词 小鼠胚胎干细胞 绿色荧光蛋白 转染 分化 心肌细胞 mouse embryonic stem cells, green fluorescence protein, transfection, differentiation, cardiomycytes
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参考文献14

  • 1Evans M J, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature, 1981, 292: 154- 156
  • 2Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. PNAS of the USA, 1981, 78:7634- 7638
  • 3Gossler A, Joyner AL, Rossant J. Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes.Science, 1989, 244:463 - 465
  • 4Klug MG, Soonpaa MH, Koh GY et al. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. The Journal of Clinical Investigation, 1996, 98:216 - 224
  • 5Eiges R, Schuldiner M, Drukker M. Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Current Biology, 2001, 11:514 - 518
  • 6赵文宁,孟国良,薛友纺.3个不同品系小鼠ES细胞的绿色荧光蛋白标记(英文)[J].Acta Genetica Sinica,2003,30(8):743-749. 被引量:1
  • 7TengL(滕路), Meng GL(孟国良), Xing Y(邢阳) et al.Labeling embryonic stem cells with enhance green fluorescent protein on the hypoxanthineguanine phosphoribosyl transferase locus.Chinese Medical Journal ( 中华医学杂志 ), 2003,116 (2): 267 -272
  • 8沈干,从笑倩,汪铮,吴春芳,曹谊林.小鼠胚胎干细胞建系及GFP标记[J].东南大学学报(医学版),2003,22(2):71-74. 被引量:6
  • 9孟国良,尚克刚.小鼠胚胎原代细胞制作方法的改进[J].生物技术,1997,7(2):38-39. 被引量:4
  • 10Sambrook J, Russell DW et al. Molecular Cloning: A Laboratory Manual. 3rd ed, New York: Cold Spring Harbor Laboratory Press,2001

二级参考文献5

共引文献8

同被引文献14

  • 1屈顺林,唐蔚青,黎健.尼克酰胺腺嘌呤二核苷酸磷酸氧化酶与动脉粥样硬化[J].中国动脉硬化杂志,2005,13(2):228-232. 被引量:11
  • 2Higuchi Y,Otsu K,Nishida K,Hirotani S,Nakayama H,Yamaguchi O,et al.Involvement of reactive oxygen species-mediated NF-kappa B activation in TNF-alpha-induced cardiomyocyte hypertrophy[J].J Mol Cell Cardiol,2002,34 (2):233-240
  • 3von Harsdorf R,Li PF,Dietz R.Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis[J].Circulation,1999,99 (22):2 934-941
  • 4Sauer H,Rahimi G,Hescheler J,Wartenberg M.Role of reactive oxygen species and phosphatidylinositol 3-kinase in cardiomyocyte differentiation of embryonic stem cells[J].FEBS Lett,2000,476 (3):218-223
  • 5Guzik TJ,West NE,Black E,McDonald D,Ratnatunga C,Pillai R,et al.Vascular superoxide production by NAD(P)H oxidase:association with endothelial dysfunction and clinical risk factors[J].Circ Res,2000,86 (9):E85-90
  • 6Babior BM.NADPH oxidase:an update[J].Blood,1999,93 (5):1 464-476
  • 7Li J,Puceat M,Perez-Terzic C,Mery A,Nakamura K,Michalak M,et al.Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis[J].J Cell Biol,2002,158 (1):103-113
  • 8Maltsev VA,Wobus AM,Rohwedel J,Bader M,Hescheler J.Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents[J].Circ Res,1994,75 (2):233-244
  • 9Irminger-Finger I,Soriano JV,Vaudan G,Montesano R,Sappino AP.In vitro repression of Brca1-associated RING domain gene,Bard1,induces phenotypic changes in mammary epithelial cells[J].J Cell Biol,1998,143 (5):1 329-339
  • 10Brubacher JL,Bols NC.Chemically de-acetylated 2',7'-dichlorodihydrofluorescein diacetate as a probe of respiratory burst activity in mononuclear phagocytes[J].J Immunol Meth,2001,251 (1-2):81-91

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