Osteogenesis and molecular mechanism of recombinant adenovirus expressing human LIM mineralization protein-1

Abstract

Objective To construct a recombinant adenovirus vector containing LIM mineralization protein-1 (LMP-1), and investigate the osteoinductive activity of bone marrow mesenchymal stem cells (BMSCs) which were transduced with recombinant adenoviral vector carrying LMP-1 gene and the molecular mechanism. Methods LMP-1 gene was acquired by polymerase chain reaction (PCR), and inserted into the pShuttle-IRES-hrGFP-1. The adenovirus Shuttle Plasmid pS-LMP-1-GFP was constructed and transformed into E. coli DH5a. Recombinant adenovirus vector carrying human LMP-1 gene (pAdLMP-1-GFP) was obtained from homologous recombination in BJ5183 cells by pS-LMP-1-GFP with plasmid pAdEasy-1. The Pac I was used to expose the long terminal repeats on both sides, and the plasmid was transfected into AD293 cells to get the adenovirus Ad-LMP-1-GFP. Ad-LMP-1-GFP was infected into the 3rd passage of BMSCs. The expression of LMP-1 was detected by RT-PCR. The osteogenic activity of BMSCs and the molecular mechanism were evaluated by the activity of alkaline phosphatase (ALP) and the expression of osteocalcin and β-catenin detected by Western blotting. Results The LMP-1 gene was successfully acquired and confirmed and packaged to recombinant adenovirus. The expression of LMP-1 was detected successfully in BMSCs. The ALP activity increased significantly, P=0.000 comparing to the control group, ALP activity were 0.096, 0.116, 0.118, 0.119 (0.110±0.011) and 0.045, 0.057, 0.056, 0.054 (0.053±0.005) respectively; the expression of osteocalcin and β-catenin enhanced significantly, P=0.001 and P=0.002 comparing to the control group, osteocalcin was 0.661, 0.767, 0.651 (0.693±0.064) and 0.177, 0.290, 0.222 (0.229±0.050); β-catenin was 0.841, 0.806, 0.867 (0.838±0.030) and 0.185, 0.236, 0.266 (0.229±0.040) respectively. Conclusion We successfully construct the Ad-LMP-1 recombinant adenovirus vector and get a high transfection efficacy in BMSCs. LMP-1 gene can induce the osteoblast differentiation of BMSCs and improve the osteogenic activity. And the β-catenin signaling molecule may play a role in this effect. Key words: LIM mineralization protein-1; Mesenchymal stem cells; Osteogenic activity; Gene transfect