Lentiviral-mediated multiple gene transfer to chondrocytes promotes chondrocyte differentiation and bone formation in rabbit bone marrow-derived mesenchymal stem cells.

Abstract

The aim of the present study was to provide a theoretical and experimental foundation on the differentiation of stem cells through the induction of multiple genes. The lentiviral vector carrying TGF-β1 and IL-10 genes was transfected to bone marrow-derived mesenchymal stem cells (BMSCs) which differentiated into chondrogenesis. Healthy New Zealand white rabbits, 2-3months of age were used in the present study. A 6-8ml of bone marrow was isolated from the iliac and tibial shaft of each rabbit. The BMSCs suspension was aspired following centrifugation of the bone marrow by percoll separating medium. The BMSCs were primarily cultured and subcultured invitro, then divided into four groups according to the difference of lentivirus vectors: groupA, receiving transforming growth factor β1 (TGF‑β1); groupB, receiving TGF-β1 and Interleukin-10 (IL-10); groupC, empty vector transfection; and groupD, receiving no cell growth factor. Fluorescence expression was detected 12h after transfecting the lentiviral vector carrying the TGF-β1 and IL-10 gene to BMSCs. The transfection efficiency was approximately 70% with a MOI=100 after 96h. Expression of SOX-9 aggrecan and TypeⅡ collagen in groupsA-E on day7 and 14 was detected by RT-PCR and western blot analysis. The expression level of three genes expressed in groupsA and C were higher compared to the expression in groupsB, D and E. The expression level of the three genes expressed in groupB was higher compared to the expression in groupD. The expression level of three genes expressed in groupA and C showed no statistical difference. Cytokines therefore play an important role in cell proliferation and chondrogenic differentiation. TGF-β1 has a synergistic effect in the differentiation. In addition, IL-10 may have a protective role in the restoration of cartilaginous tissue.