Experimental study on effects of LIM mineralization protein-1 on the osteogenic potential of cultured bone marrow mesenchymal stem cells

Abstract:

Objective：To study influences of LIM mineralization protein（ＬＭＰ－１） on the osteogenic potential of cultured bone marrow mesenchymal stem cells（BMSCs） by constructing eukaryotic expression plasmid carrying LMP-1 and having it expressed in rabbit BMSCs. Methods：Reverse transcription- polymerase chain reation（RT-PCR） technique was used to clone hLMP-1 and hBMP-2 from human placenta tissue and plasmid with hBMP-2. Digestion and ligations with eukaryotic expression plasmid pIRES2-EGFP，pIRES2-EGFP-LMP-1 and pIRES2-EGFP-BMP-2 recombinants were constructed. BMSCs were isolated and culture-expanded from rabbit bone marrow. The third culture was chosen for study. With the help of lipofectamine，plasmids pIRES2-EGFP-LMP-1，pIRES2-EGFP-BMP-2 and pIRES2-EGFP were transfected into BMSCs. Transfective rate was estimated under fluorescence microscope and cells were tested seven days later. Expressions of hLMP-1，hBMP-2 and collagen type Ⅰ were tested with RT-PCR. Alkaline phosphatase（ALP） testing kit was used to measure ALP activity and immunohistochemical staining was used to test the expression of osteocalcin. Results：Restriction and sequencing analyses confirmed that eukaryotic expression plasmid pIRES2-EGFP-LMP-1 and pIRES2-EGFP-BMP-2 was successfully constructed. 15% transfection rate was estimated by fluorescence microscope. Groups transfected with pIRES2-EGFP-LMP-1 and pIRES2-EGFP-BMP-2 expressed hLMP-1 and hBMP-2. Both groups enhanced the activity of ALP and induced the expression of collagen type Ⅰ and osteocalcin. Conclusion：LMP-1 can promote the osteogenic potential of cultured BM－SCs，which lays a foundation for gene therapy with LMP-1.