Construction of recombinant adenovirus expressing combined human hypoxia inducible factor-1α and LIM mineralization protein-1 and its osteogenic effect

Objective This study was designed to construct a recombinant adenovirus vector expressing both hypoxia inducible factor-1α (HIF-1α) and LIM mineralization protein-1 (LMP-1) gene linked by internal ribozyme entry site (IRES), and to detect the osteogenesis in rat bone marrow stromal stem cells. Methods The full-length of human HIF-1α (2 481 bp) and LMP-1 (1 374 bp) genes and IRES (564 bp) sequence were acquired by polymerase chain reaction (PCR) and inserted into pShuttle-IRES-hrGFP-1 to construct recombinant adenovirus shuttle plasmid pS-HIF-LMP-1-GFP. Recombinant adenovirus vector pAd-HIF-LMP-1-GFP was obtained from homologous recombination in BJ5183 cells by pS-HIF-LMP-1-GFP and pAdEasy-1. Recombinant adenovirus Ad-HIF-LMP-1-GFP was obtained by packaging in AD293 cells. Ad-HIF-LMP-1-GFP was transduced to the rat bone marrow mesenchymal stem cells (BMSCs). The expression of HIF-1α and LMP-1 in BMSCs was observed by reverse transcription-polymerase chain reaction (RT-PCR). The alkaline phosphatase (ALP) activity, calcium formation, osteocalcin and related transcription factor-2 (Runx2) expression changes between experimental group and control group were detected to evaluate the osteogenitic capacity of combined HIF-1α and LMP-1. Results The recombinant adenovirus expressing combined HIF-1α and LMP-1 was successfully acquired. The expression of HIF-1α and LMP-1 was detected successfully in BMSCs. The ALP activity increased significantly (P=0.000). ALP activity was 0.145, 0.160, 0.170, 0.167 (0.160±0.011) ng/μg protein in experimental group and that was 0.045, 0.057, 0.056, 0.054 (0.053±0.005) ng/μg protein in control group, respectively. The calcium formation increased significantly (P=0.001). The quantification was 0.636, 0.544, 0.591, 0.515 (0.570±0.050) in experimental group and that was 0.121, 0.150, 0.144, 0.167 (0.145±0.019) in control group, respectively. The expression of osteocalcin and Runx2 was enhanced significantly (P=0.017 and 0.000). The expression of osteocalcin was 2.688, 2.997, 2.777 (2.820±0.159) in experimental group and that was 2.009, 1.916, 1.944 (1.956±0.047) in control group, respectively. The expression of Runx2 was 0.440, 0.465, 0.456 (0.454±0.010) in experimental group and that was 0.096, 0.108, 0.117 (0.107±0.010) in control group, respectively. Conclusion The recombinant adenovirus vector was successfully constructed by using IRES sequence to connect human HIF-1 alpha and LMP-1 gene. Combined HIF-1α and LMP-1 gene can promote the osteoblastic differentiation of BMSCs. Key words: Hypoxia inducible factor-1α; LIM mineralization protein-1; Mesenchymal stem cells; Osteogenic activity; Gene transfection