Abstract:

BACKGROUND: Studies shows that the LIM mineralization protein 1 (LMP-1) can stimulate bone formation in vivo and in vitro.
OBJECTIVE: To construct the recombinant adenovirus vector containing LMP-1 gene by using the Ad-Easy system, then to detect the gene expression and study the mechanism of bone formation induced by LMP-1 in infected dog bone marrow mesenchymal stem cells (BMSCs).
METHODS: The recombinant plasmid pAd-LMP-1 was constructed. The replication-defective recombinant adenovirus Ad-LMP-1 was packaged and amplified in the human embryonic kidney 293 cells (HEK293). Dog BMSCs were infected by Ad-LMP-1 in the best value of MOI in vitro. The gene expression of LMP-1and BMP-7 was detected by RT-PCR and Western Blot. Dogs BMSCs were treated with Ad-LMP-1 and recombinant human bone morphogenetic protein 7 (rhBMP-7) to study the mechanism of bone formation of LMP-1 undergoing mineralization (Ca) nodules at 21 days by alizarin red staining.
RESULTS AND CONCLUSION: ①Ad-LMP-1 was successfully constructed and obtained titers in the packaging about 1×10¹¹ efu/mL after amplified in HEK293. ②The best efficiency could be obtained at the most appropriate MOI 100 after Ad-LMP-1 infected BMSCs. Infected BMSCs can express mRNA and protein of LMP-1, but not caused expression of BMP-7. ③ Dog BMSCs were treated with Ad-LMP-1 and (or) rhBMP-7 for 21 days. We found that Ad-LMP-1 with exogenous rhBMP-7 promote transformation into osteoblasts together. Neither Ad-LMP-1 nor rhBMP-7 alone induced any transformation on day 21. The recombinant adenoviral vector containing hLMP-1 gene was successfully constructed and expressed effectively in dogs BMSCs. It is confirmed that the mechanism of bone formation induced by LMP-1 is synergistic effect with the performance of exogenous rhBMP-7.