BACKGROUND: Whether bone morphogenetic protein 2 (BMP-2) can be transduced into marrow stromal cells (MSCs) and produce osteogenic effects by viral or non-viral vector remains unclear? OBJECTIVE: To observe the expression of cultured rabbit MSCs transfected with pcDNA3-hBMP2 in vitro. Simultaneously, the MSCs were transfected but not screened and then transplanted into autologous muscle to investigate the osteogenic capability by X-ray. DESIGN, TIME AND SETTING: A controlled observation experiment was performed at the Department of Orthopedics, Liaoning Medical University between November 2004 and April 2005. MATERIALS: Six adult New Zealand rabbits, of either gender, weighing 2.0-3.0 kg, were included for this study. BMP2 antibody was the product of Sanaka Company, USA. pcDNA3-hBMP2 was provided by Professor Pu Qin from the Department of Biochemistry, Fourth Military Medical University of Chinese PLA). Restriction enzyme was purchased from Takara biotechnology (Dalian) CO., LTD., China. METHODS: Super-purified plasmid pcDNA3-hBMP2 was extracted from E. coli. Bone marrow was taken from the adult rabbit femur for harvesting MSCs by density gradient separation. The MSCs were divided into the following 4 groups: Group A, cells were transfected and screened by G418; Group B, cells were transfected by pcDNA3-hBMP2; Group C, cells were transfected by empty vector pcDNA3; Group D, only transfection reagent Fugene 6 was added.MAIN OUTCOME MEASURES: Transient BMP2 expression was analyzed by immunohistochemistry. Expression of osteocalcin and collagen I was examined by immunohistochemistry and in situ hybridization, respectively. Two weeks after transfection, MSCs from the group B were autologously transplanted into the muscle. Four weeks later, X-ray assay was used to observe bone formation.RESULTS: pcDNA3-hBMP2 was successfully transduced into MSCs and transiently expressed BMP2 100%. Four weeks after gene transfection, expression levels of osteocalcin and collagen I were significantly higher in the group A than in the groups C and D. X-ray results demonstrated new bone formation four weeks after MSCs transplanted into the muscle.CONCLUSION: pcDNA3-hBMP2 can safely and efficiently transfect MSCs and induce them to differentiate towards osteoblasts by secreting BMP2.

Objective To examine the therapeutic effect of C Ⅱ TA inhibition in collagen-induced arthritis(CIA),using a delivery system tailored to target C ⅡTA gene by small interfering RNA (siRNA).Methods Mice with collagen-induced arthritis were injected intravenously with C Ⅱ TA siRNA.The clinical score was monitored for up to 4 weeks after treatment.The severity of inflammation of mouse joint was evaluated by histological examination.Real-time PCR was used to determine the cytokine mRNA expression.Cytokine production was measured by ELISA from serum.T cell proliferation was examined by MTT method.Results IFN-γ and IL-17 were elevated in CIA mice,but were iuhibited significantly by C Ⅱ TA siRNA either prevention or intervention of autoimmune arthritis.Collagen specific T cell proliferation was significantly suppressed.Increased level of IL-4 by T cells was observed in C Ⅱ TA siRNA treated group compared with that of control group.Conclusion Our findings indicate that systemic RNAi-mediated C Ⅱ TA gene silencing is effective in the treatment of CIA and regulateds the balance of Th1/Th2 differentiation.