LIM矿化蛋白-1对间充质干细胞成骨分化影响的实验研究

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

目的:构建LIM矿化蛋白-1(LIM mineralization protein-1,LMP-1)的真核表达质粒,并在兔骨髓间充质干细胞(bonemarrow mesenchymal stem cells,BMSCs)内表达,研究LMP-1对体外培养BMSCs成骨分化的影响。方法:RT-PCR克隆hLMP-1和骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2),并构建pIRES2-EGFP-LMP-1及pIRES2-EGFP-BMP-2重组质粒。分离培养BMSCs,脂质体介导下将pIRES2-EGFP-LMP-1、pIRES2-EGFP-BMP-2和pIRES2-EGFP质粒转染BMSCs。7 d后收集细胞,RT-PCR比较hLMP-1、hBMP-2及Ⅰ型胶原的表达,检测碱性磷酸酶(alkaline phosphatase,ALP)活性,免疫组化染色比较骨钙素表达。结果:pIRES2-EGFP-LMP-1和pIRES2-EGFP-BMP-2重组质粒构建成功。重组质粒成功转染BMSCs,RT-PCR证实表达目的基因,hLMP-1和hBMP-2转染组可以明显促进细胞分泌ALP的活性,诱导Ⅰ型胶原和骨钙素的表达。结论:LMP-1可以促进BMSCs向成骨细胞分化。

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,pIRES2EGFP-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-EGFPBMP-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-BMP2 was successfully constructed.15% transfection rate was estimated by fluorescence microscope.Groups transfected with pIRES2EGFP-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 BMSCs,which lays a foundation for gene therapy with LMP-1.