title: [CONSTRUCTION AND IDENTIFICATION OF ADENOVIRUS VECTOR EXPRESSING BONE MORPHOGENETIC PROTEIN 2 AND TRANSFORMING GROWTH FACTOR β3 GENES AND THEIR EXPRESSION IN BONE MARROW MESENCHYMAL STEM CELLS OF DIANNAN SMALL-EAR PIGS].
abstract: OBJECTIVE
  To construct and identify the recombinant adenovirus vector expressing bone morphogenetic protein 2 (BMP-2) and transforming growth factor β3 (TGF-β3) genes, to observe the expressions of BMP-2 and TGF-β3 after transfected into bone marrow mesenchymal stem cells (BMSCs) of the Diannan small-ear pigs.
  
  
  METHODS
  BMP-2 cDNA and TGF-β3 cDNA were amplified by PCR, and were subcloned into the pEC3.1 (+) plasmid to obtain pEC-GIE 3.1-BMP-2 and pEC-GIE3.1-TGF-β3 plasmid respectively. They were subcloned into pGSadeno vector by homologous recombination reaction and HEK293 cells were transfected after linearization to obtain Ad-BMP-2 and Ad-TGF-β3. The BMSCs were isolated from the bone marrow of Diannan small-ear pig and cultured. The 3rd passage BMSCs were transfered with Ad-BMP-2 (group A), Ad-TGF-β3 (group B), Ad-BMP-2+ Ad-TGF-β3 (group C), and untransfected cells served as a control (group D). The expressions of BMP-2 and TGF-β3 genes and proteins were detected by PCR, immunofluorescence, and Western blot. The chondrogenic differentiation of BMSCs was evaluated by immunohistochemical of collagen type II.
  
  
  RESULTS
  The Ad-BMP-2 and Ad-TGF-β3 were constructed successfully and confirmed by PCR and sequencing. The expression clones of Ad-BMP-2 and Ad-TGF-β3 were packaged into maturated adenovirus successfully, the titer was 5.6 x 10(8) and 1.6 x 10(8) pfu/mL respectively. The PCR results showed a light band at 310 bp in group A and at 114 bp in group B, and both 310 bp and 114 bp bands in group C, but no band in group D. The image of immunofluorescence showed that there were red fluorescence and green fluorescence expressions in the cytoplasm of BMSCs at 72 hours after transfection in groups A and B, respectively; in group C, both red and green fluorescence expressions were detected, and no red or green fluorescence was detected in group D. The results of Western blot showed that there was a light band at 18 x 10(3) in group A and at 50 x 10(3) in group B; both 18 x 10(3) and 50 x 10(3) bands were detected in group C; but no band was detected in group D. The cells were positive for collagen type II in groups A, B, and C; group C acquired strong collagen type II staining when compared with group A and group B; in group D, the cells were negative for collagen type II staining.
  
  
  CONCLUSIONS
  The recombinant adenovirus vector expressing BMP-2 and TGF-β3 are constructed successfully. The BMP-2 and TGF-β3 genes could be expressed effectively in BMSCs of Diannan small-ear pig after transfection, which could afford modified seeding cells for cartilage tissue engineering.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.forename: Yanlin
authors.initials: Y
authors.lastname: Li
authors.forename: Yaofen
authors.initials: Y
authors.lastname: Jin
authors.forename: Jianming
authors.initials: J
authors.lastname: Chen
authors.forename: Huijian
authors.initials: H
authors.lastname: Wang
authors.forename: Chuan
authors.initials: C
authors.lastname: He
authors.forename: Shuhai
authors.initials: S
authors.lastname: Cao
authors.forename: Fengkai
authors.initials: F
authors.lastname: Zhao
mesh_terms: D000256:Adenoviridae; D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism*; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics*; D002454:Cell Differentiation; D024043:Collagen Type II; D005822:Genetic Vectors; D057809:HEK293 Cells; D006412:Hematopoietic Stem Cells; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D023822:Tissue Engineering; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics*; D053782:Transforming Growth Factor beta3 / Q000235:genetics* / Q000378:metabolism
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D024043:Collagen Type II; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D053782:Transforming Growth Factor beta3; C499469:recombinant human bone morphogenetic protein-2