Experimental Study On The Proliferation And Differentiation In Vitro Of HBMSc Transfected Of VEGF₁₆₅ And EGFP Genes By Ad Mediated

[Objective] (1) To construct and identify Ad- VEGF₁₆₅. (2) To culture and expand hBMSc in vitro and to observe the growth and biological specialties of primary and sub cultured cells. (3) To explore the possibility of stable expression of VEGF₁₆₅ and eGFP in hBMSc. (4) To observe the influences of transfection on the proliferation and differentiation of hBMSc. (5) To observe the efficiency of eGFP expression and the tracing of VEGF₁₆₅ expression. (6) To observe the effects on the osteogenesis and angiogenesis of hBMSc transfected by VEGF₁₆₅ gene in vivo in athymic mouse.[Methods] (1) VEGF₁₆₅ gene was synthesized with PCR and cloned with pMD18-T vector. The cloned VEGF₁₆₅ cDNA fragments were sequenced and done NCBI BLAST similar analysis. The pMD18-T-VEGF₁₆₅ and empty plasmid pDNR-Ir was digested by EcoR I and BamH I, ligated and transformed competence E.coli DH5a. Positive recombinants were identified by EcoR I and Bam H I digestion. The plnt.AVI.SpaT-VEGF₁₆₅ was synthesized by homologous recombination of pDNR-VEGF₁₆₅ and pInt.AV I.SpaT with Cre-loxP system and identified by EcoR I digestion. Replication defective adenovirus with VEGF₁₆₅ gene was synthesized by coinfection of pInt.AV I.SpaT VEGF₁₆₅ and plnt.B.B ptasmids into 293 cells. Cytopathogenic effects were observed through invert microscope. Adenovirus DNA was extracted from cytopathogenic 293 cell lysis, VEGF₁₆₅ gene was identified with PCR. The recombinant adenovirus titers were tested with median tissue culture infective dose (TCID50) method. (2) Bone marrow was aspirated from the dead...