Osteogenic differentiation of adipose-derived stem cells transfected by lentivirus vector carrying LIM mineralization protein-1 and hypoxia-inducible factor-1alpha / 中国组织工程研究

ABSTRACT

BACKGROUND:LIM mineralization protein-1 (LMP-1) and hypoxia-inducible factor-1α (HIF-1α) as intracelular proteins can induce osteogenic differentiation and promote angiogenesis, respectively. Therefore, their combination is of great significance for effectively inducing the osteogenic differentiation of adipose-derived stem cels. OBJECTIVE:To study the osteogenic differentiation of adipose-derived stem cels transfected by lentivirus vector carrying LMP-1 and HIF-1α. METHODS:Reverse transcription-PCR technology was employed to clone LMP-1 and HIF-1α genes, and thegenes were cloned to lentivirus vectors pLVX-EF1α-DsRed-Hyg and pLVX-EF1α-IRES2-AcGFP1 to construct main lentiviral vectors pLVX-EF1α-DsRed-Hyg-RLMP-1 and pLVX-EF1α-IRES2-AcGFP1-RHIF-1α. Then, Lenti-X 293T cels were transfected with main vectors pLVX-EF1α-DsRed-Hyg-RLMP-1 and pLVX-EF1α-IRES2-AcGFP1-RHIF-1α, packaging plasmid and coated plasmid. After that, lentiviral vectors were packaged to transfect adipose-derived stem cels from rats that were obtained by tissue explants culture and enzyme digestion methods. At 3, 7, 14 days after transfection, reverse transcription-PCR technology was adopted to detect the expression of osteogeic genes, such as bone morphogenetic protein 2, Runx-2, alkaline phosphatase, osteocalcin as wel as to detect the expression of vascular endothelial growth factor. RESULTS AND CONCLUSION:Lentiviral vectors pLVX-EF1α-DsRed-Hyg-RLMP-1 and pLVX-EF1α-IRES2-AcGFP1-RHIF-1α were effectively transfected into adipose-derived stem cels. Reverse transcription-PCR results showed that from the 7th day to the 14th day after lentivirus transfection, bone morphogenetic protein 2, Runx-2, alkaline phosphatase and osteocalcin al over-expressed. These findings indicate that the combination of LMP-1 and HIF-1α can enhance the osteogenic activity of adipose-derived stem cels.