title: [Effect of GFP-containing lentivirus infection on the expression of octamer transcription factor 4 in human umbilical cord mesenchymal stem cells].
abstract: OBJECTIVE
  To infect human umbilical cord mesenchymal stem cells (hUCMSCs) with lentivirus carrying green fluorescence protein (GFP) and observe its effect on octamer binding transcription factor 4 (Oct4) expression.
  
  
  METHODS
  Mesenchymal stem cells isolated from human umbilical cord by tissue adherence method were cultured and detected for the the surface markers by flow cytometry. HUCMSCs were infected with lentivirus carrying GFP at different multiplicity of infection (MOI). GFP expression efficiency was observed using the fluorescence microscope and flow cytometry to obtain optimal MOI value. The experiment included non-transfection group and the GFP transfection group. The effect of GFP lentivirus on cell proliferation was evaluated by MTT. Oct4 expression in hUCMSCs cultured in vitro continuously for 2 and 8 weeks was measured by quantitative reverse transcriptase PCR (qRT-PCR) and immunofluorescence staining.
  
  
  RESULTS
  HUCMSCs displayed fusiform shape as fibroblasts in vitro. Flow cytometry revealed that the third-passage cells highly expressed CD29, CD105 and CD90, and lowly expressed CD34 and CD45. At 96 h after infection with GFP lentivirus (MOI=20), GFP positive rate of hUCMSCs was more than 75%, the highest level. MTT showed nearly no change in cell proliferation in the transfected group in comparison with the non-transfection group (P>0.05). The relative expression of Oct4 detected by qRT-PCR in cells cultured for 2 and 8 weeks were 0.9075±0.0124 and 0.8600±0.0135, respectively. Immunofluorescence staining showed that Oct4 expression was localized in the nucleus of cells.
  
  
  CONCLUSIONS
  Lentivirus-mediated GFP gene can be expressed in hUCMSCs and has no significant effect on the expression of Oct4.
authors: Department of Cardiology, Chongqing Medical University, Chongqing, China.
authors: Li
authors: L
authors: Tang
authors: Jing
authors: J
authors: Chang
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D005260:Female; D015870:Gene Expression; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D007231:Infant, Newborn; D019012:Integrin beta1 / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000235:genetics*; D011247:Pregnancy; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D018800:Thy-1 Antigens / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D014470:Umbilical Cord / Q000166:cytology*
chemical_list: D019012:Integrin beta1; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D047888:Receptors, Tumor Necrosis Factor, Type I; D018800:Thy-1 Antigens; D049452:Green Fluorescent Proteins