LIM矿化蛋白-1对间充质干细胞成骨分化影响的实验研究
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Experimental study on effects of LIM mineralization protein-1 on the osteogenic potential of cultured bone marrow mesenchymal stem cells
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    摘要:

    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(bone marrow 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。

    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.

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徐希彦,邓 毅,李修洋,邓忠良. LIM矿化蛋白-1对间充质干细胞成骨分化影响的实验研究[J].重庆医科大学学报,2013,(1):14-19

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  • 在线发布日期: 2013-01-08
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