Experimental study on effects of LIM mineralization protein-1 on the osteogenic potential of cultured bone marrow mesenchymal stem cells
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Experimental study on effects of LIM mineralization protein-1 on the osteogenic potential of cultured bone marrow mesenchymal stem cells
Journal of Chongqing Medical UniversityVol. 36, Issue 1, Pages: 14-19(2013)
作者机构:
1. 重庆医科大学附属第二医院骨科,重庆,400010
2. 重庆市中医骨科医院骨科,重庆,400010
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Published:2013,
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XU Xiyan, DENG Yi, LI Xiuyang, et al. Experimental study on effects of LIM mineralization protein-1 on the osteogenic potential of cultured bone marrow mesenchymal stem cells. [J]. Journal of Chongqing Medical University 36(1):14-19(2013)
DOI:
XU Xiyan, DENG Yi, LI Xiuyang, et al. Experimental study on effects of LIM mineralization protein-1 on the osteogenic potential of cultured bone marrow mesenchymal stem cells. [J]. Journal of Chongqing Medical University 36(1):14-19(2013)DOI:
Experimental study on effects of LIM mineralization protein-1 on the osteogenic potential of cultured bone marrow mesenchymal stem cells
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.
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