Establishment of human mesenchymal stem cells transferred by vascular endothelial growth factor / 中国组织工程研究

ABSTRACT

BACKGROUND: Vascular endothelial growth factor (VEGF) is able to effectively treat the ischemic heart disease, but in vivo VEGF cannot be maintained effective concentration. OBJECTIVE: To detect the expression of VEGF mRNA and protein in human bone marrow mensenchymal stem cells transferred by VEGF-165 gene. DESIGN, TIME AND SETTING: The empirical study was conducted from March 2006 to April 2007 at Shanghai Chest Hospital. MATERIALS: Human bone marrow mesenchymal stem cell line and VEGF were offered by Basic Laboratory, Thoracic Tumor Institute, Shanghai Chest Hospital. METHODS: hVEGF165 gene was reconstructed in pcPGK-vector and transferred into human bone marrow mensenchymal stem cells (BMSCs) by liposome-mediated method, clone screening by G418. MAIN OUTCOME MEASURES: The mRNA and protein of VEGF gene in transferred cells was detected by reverse transcription-polymerase chain reaction (RT-PCR), Real time PCR, Western Blot, enzyme linked immunosorbent assay (ELISA) in 3 stem cells of pcPGK-VEGF165-IRES-GFP and pcPGK- IRES-GFP, respectively. RESULTS: pcPGK-hVEGF165 vector was reconstructed and transferred into hMSCs successfully. The expression of hVEGF165 in the transfected hMSCs was demonstrated with RT-PCR and Real time PCR. Western Blot and ELISA demonstrated that the expression of hVEGF165 in the transfected hMSCs and VEGF protein in supernatant were significantly more than untransfected hMSCs. CONCLUSION: hVEGF165 can be successfully transfected into BMSCs by using liposome mediated gene transfer. Stably expressed VEGF165 cell line can be obtained..