title: [Recombinant adenovirus Ad-human matrix metalloproteinase 1 transfecting bone marrow mesenchymal stem cells of rats in vitro].
abstract: OBJECTIVE
  To transfect bone marrow mesenchymal stem cells (BMSCs) of rats by recombinant adenovirus Ad-human matrix metalloproteinase 1 (hMMP-1) in vitro so as to lay the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation.
  
  
  METHODS
  BMSCs were isolated from bone marrow of 2-3 weeks old Sprague Dawley rats by whole bone marrow adherence method and identified, then transfected by recombinant adenovirus Ad-hMMP-1 carrying enhanced green fluorescent protein (EGFP) marker in vitro. The green fluorescent expression was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry to determine the optimum multiplicity of infection (MOI). BMSCs at passage 3 were divided into 3 groups: untransfected BMSCs group (group A), Ad-EGFP transfected BMSCs group (group B), and Ad-hMMP-1-EGFP transfected BMSCs group (group C); the gene and intracellular protein of hMMP-1 were detected by RT-PCR and Western blot; the ELISA assay was used to detect the supernatant protein expression, and the hMMP-1 activity was measured by fluorescent quantification kit.
  
  
  RESULTS
  The green fluorescent was observed in BMSCs transfected by recombinant adenovirus at 24 hours after transfection; the fluorescence intensity was highest at 72 hours; and the optimum MOI was 200. The cells of 3 groups entered the logarithmic growth phase on the 3rd day and reached plateau phase on the 6th day by MTT assay; no significant difference was found in the cell proliferation rate among 3 groups (P > 0.05). RT-PCR, Western blot, and ELISA assay showed high expressions of the hMMP-1 gene and protein in group C, but no expression in groups A and B. The hMMP-1 activity was 1.24 nmol/(mg.min) in group C, but hMMP-1 activity was not detectable in groups A and B.
  
  
  CONCLUSIONS
  The exogenous hMMP-1 gene is successfully transfected into BMSCs of rats via recombinant adenovirus and can highly express, which lays the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation.
authors.affiliation: Department of Postgraduate, Third Military Medical University, Chongqing, 400038, P.R.China.
authors.forename: Chao
authors.initials: C
authors.lastname: Du
authors.forename: Mingde
authors.initials: M
authors.lastname: Jiang
authors.forename: Weizheng
authors.initials: W
authors.lastname: Zeng
authors.forename: Shumei
authors.initials: S
authors.lastname: Zheng
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Wei
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000235:genetics* / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D023822:Tissue Engineering; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C522360:MMP1 protein, human; D020781:Matrix Metalloproteinase 1