title: [Osteodifferentiation of bone marrow mesenchymal stem cells after transfected by lentiviral vector mediated bone morphogenetic protein 2].
abstract: OBJECTIVE
  To construct recombinant lentiviral vectors of porcine bone morphogenetic protein 2 (BMP-2) gene and to detect BMP-2 gene activity and bone marrow mesenchymal stem cells (BMSCs) osteogenetic differentiation so as to lay a foundation of the further study of osteochondral tissue engineering.
  
  
  METHODS
  BMSCs were isolated from bone marrow of 2-month-old Bama miniature porcines (weighing, 15 kg), and the 2nd generation of BMSCs were harvested for experiments. The porcine BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and was used to transfect BMSCs at multiplicity of infection (MOI) of 10, 25, 50, 100, and 200, then the optimal value of MOI was determined by fluorescent microscope and inverted phase contrast microscope. BMSCs transfected by BMP-2 recombinant lentiviral vectors served as experimental group (BMP-2 vector group); BMSCs transfected by empty vector (empty vector group), and non-transfected BMSCs (non-transfection group) were used as control groups. RT-PCR, immunohistochemistry staining, and Western blot were performed to detect the expressions of BMP-2 mRNA and protein. Then the BMSCs osteogenesis was detected by alkaline phosphatase (ALP) staining, ALP activities, and Alizarin red staining.
  
  
  RESULTS
  The recombinant lentiviral vectors of porcine BMP-2 gene was successfully constructed and identified by RT-PCR and gene sequencing, and BMSCs were successfully transfected by BMP-2 recombinant lentiviral vectors. Green fluorescent protein could be seen in the transfected BMSCs, especially at MOI of 100 with best expression. The immunohistochemistry staining and Western blot showed that BMSCs transfected by BMP-2 recombinant lentiviral vectors could express BMP-2 protein continuously and stably at a high level. After cultivation of 2 weeks, the expression of ALP and the form of calcium nodules were observed.
  
  
  CONCLUSIONS
  The porcine BMP-2 gene lentiviral vector is successfully constructed and transfected into the BMSCs, which can express BMP-2 gene and protein continuously and stably at a high level and induce BMSCs differentiation into osteoblasts.
authors: Department of Orthopedics, Mindong Hospital Affiliated to Fujian Medical University, Ningde Fujian, 355000, P.R.China.
authors: Shenggui
authors: S
authors: Xu
authors: Department of Orthopedics, the First Affiliated Hospital of Fujian Medical University.
authors: Jianhua
authors: J
authors: Lin
authors: Department of Orthopedics, the Affiliated People's Hospital of Fujian University of Traditional Chinese Medicine.
authors: Weinan
authors: W
authors: Liu
authors: Department of Orthopedics, the First Affiliated Hospital of Fujian Medical University.
authors: Chaoyang
authors: C
authors: Wu
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D010012:Osteogenesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D023822:Tissue Engineering / Q000379:methods; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism
chemical_list: D055396:Bone Morphogenetic Protein 2; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D000469:Alkaline Phosphatase