重组pcDNA3.1hBMP7真核表达载体的构建及转染兔骨髓基质干细胞的实验研究.DOCVIP

临床医学论文-重组pcDNA3.1hBMP7真核表达载体的构建及转染兔骨髓基质干细胞的实验研究 【摘要】? 构建人骨形态发生蛋白-7(human bone morphogenetic protein7，hBMP7)基因真核表达载体，评价其转染兔骨髓基质干细胞(bone marrow stromal cells,BMSCs)的诱导成骨能力。［方法］从人类胎盘组织中克隆出hBMP7基因，与真核表达载体pcDNA3.1连接，成功构建了重组pcDNA3.1hBMP7真核表达载体；从兔骨髓中分离培养BMSCs，分为3组：A pcDNA3.1hBMP7转染组；B空载体pcDNA3.1转染组；C未转染组。使用RTPCR、免疫组织化学等方法检测hBMP7在BMSCs中的表达。检测各组细胞碱性磷酸酶，胶原，骨钙蛋白合成情况。［结果］RTPCR、免疫组织化学检测显示经hBMP7转染的BMSCs中有BMP7表达。经hBMP7转染BMSCs的碱性磷酸酶于转染后第2 d显著增高，到第8 d达到最高;经hBMP7转染的BMSCs合成胶原、骨钙蛋白能力也显著提高，与转染空载体、未转染的BMSCs相比有显著性差异(P0.05)。［结论］成功构建了pcDNA3.1hBMP7真核表达载体;外源的hBMP7基因可在兔BMSCs中充分、高效的表达，并且这种外源性hBMP7基因具备了促进兔骨髓基质干细胞向成骨细胞转化的能力，这为hBMP7的基因治疗提供了坚实的理论基础。 【关键词】? 人骨形态发生蛋白7 基因转染 骨髓基质干 ??? Abstract：［Objective］To construct arecombinant eukaryotic expression plasmid carrying human BMP7 gene which was transfected into bone marrow stromal cells (BMSCs) in vitro and expressed.［Method］Clone human BMP7 cDNA from a Chinese woman placenta was recombinanted with plasmid pcDNA3.1, to construct eukaryotic expression carrier of recombinaut pcDNA3.1hBMP-7.The BMSCs were isolated from rabbit bone marrow and cultured in vitro.They were divided into three groups: pcDNA3. 1hBMP7 transduced group; pcDNA3.1 transduced group;untransduced group.The expression of hBMP7 was detected by RTPCR,Immuneohistochemistry. ALP, Collogen, Osteocalcin production were detected.［Result］The pcDNA3.1hBMP7 transduced BMSCs expressed hBMP7 at both mRNA and protein levels.ALP activity was detected in pcDNA3.1hBMP7 transduced cells from day 2 to day 10,? peaking on day 8. Collogen,? osteocalcin production were elevated significantly too (P0.05).［Conclusion］Eukaryotic expression carrier of recombinant pcDNA3.1BMP7 was constructed successfully.The results indicate that hBMP7 is expressed in BMSCs sufficiently and is involved in inducing differentiation of BMSCs into osteoblast.The method would provide substantial basement for hBMP7 gene therapy. ??? Key words：hBMP7;? genes transfection;? bone marrow stromal cell