LIM矿化蛋白-1（LMP-1）对骨髓间充质干细胞成骨分化影响的实验研究

发布时间：2018-08-12 11:28

【摘要】： 目的:通过构建LIM矿化蛋白-1(LMP-1)的真核表达质粒,并使其在分离培养的兔骨髓间充质干细胞(BMSCs)内表达,研究LMP-1对体外培养BMSCs成骨分化的影响。 方法:采用RT-PCR的方法分别从人的胎盘组织和含有人BMP-2的重组质粒中,克隆LMP-1和BMP-2,与真核表达质粒pIRES2-EGFP酶切连接,构建pIRES2-EGFP-LMP1及pIRES2-EGFP-BMP2重组质粒。采用梯度离心方法从兔骨髓中分离培养BMSCs。以第三代细胞为实验对象,脂质体介导下分别将pIRES2-EGFP-LMP1、pIRES2-EGFP-BMP2和pIRES2-EGFP质粒转染细胞。荧光显微镜下观察转染效率,7天后收集细胞检测其生物学性状的变化。RT-PCR比较LMP-1、BMP-2及Ⅰ型胶原的表达,碱性磷酸酶活性检测ALP活性,免疫组化染色比较骨钙素表达。 结果:经酶切和测序鉴定pIRES2-EGFP-LMP1和pIRES2-EGFP-BMP2重组质粒构建成功。成熟的体外分离培养BMSCs方法方便简单,收获细胞数量多且增殖能力强。脂质体介导重组质粒转染BMSCs后,通过荧光显微镜观察转染成功且转染效率15%左右。收集细胞RT-PCR证实表达目的基因,LMP-1和BMP-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。
[Abstract]:Aim: to construct eukaryotic expression plasmid of LIM mineralized protein 1 (LMP-1) and express it in (BMSCs) of cultured rabbit bone marrow mesenchymal stem cells (BMSCs), and to study the effect of LMP-1 on osteogenic differentiation of BMSCs cultured in vitro. Methods: LMP-1 and BMP-2 were cloned from human placental tissues and recombinant plasmids containing human BMP-2 by RT-PCR and ligated with eukaryotic expression plasmid pIRES2-EGFP to construct pIRES2-EGFP-LMP1 and pIRES2-EGFP-BMP2 recombinant plasmids. BMSCs were isolated from rabbit bone marrow by gradient centrifugation. The pIRES2-EGFP-LMP1 pIRES2-EGFP-BMP2 and pIRES2-EGFP plasmids were transfected into the cells by liposome-mediated transfection. The expression of LMP-1BMP-2 and collagen 鈪，

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