Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells to Myogenic Cells and Expression of VEGF After Gene Transfection

摘要

Objectives To induce thedifferentiation of rabbit bone marrow mesenchymal stemcells (MSCs) to myogenic cells in vitro, and toinvestigate the expression of vascular endothelial growthfactor (VEGF) gene in MSCs transfected byAdTrackCMV-VEGF165. Methods MSCs wereisolated and purified from rabbit bone marrow by percoll(1.073 g /ml) and then cultured in low glucose DMEMwith 10% FBS. AdTrackCMV-VEGF165 eukaryoticexpression vector was constructed and transfected intothe MSCs. After being incubated with 5-azacytidine (5-Aza), the expression of troponin I in MSCs was assayedby immunohistochemistry and the expression of VEGFgene was identified by northern blot and western blot.The concentration of VEGF in the supernatant wasmeasured by ELASA. Results MSCs were isolatedand cultured successfully from rabbit bone marrow. Thepositive cTnI stain of some MSCs after the induction of5-aza indicated that the cells were differentiated tomyogenic cells. Northern blot and western blot showedthat the expression of VEGF 165 mRNA was muchhigher in the hVEGF165 gene transfected cells thanthat of the control. The concentration of VEGF in thesupernatant got to the peak 3-5 days after hVEGF165gene transfection (1011- 1027 pg/ml) and decreasedgradually thereafter, but still higher than that of controlgroup or pAdTrackCMV group (349 pg/mL vs 116 pg /mLor 125pg/ml respectively, P< 0.01). ConclusionsMSCs could be induced to differentiate to myogeniccells by 5-aza in vitro and could express VEGF byVEGF gene transfection.