骨形成蛋白7；基因转染；软骨细胞_

重组人BMP7真核表达质粒的构建及转染软骨细胞后的表达

http://www.100md.com 《中国矫形外科杂志》 2006年第11期

骨形成蛋白7；基因转染；软骨细胞,,骨形成蛋白7；,基因转染；,软骨细胞,1材料与方法,2结果,3讨论,参考文献：

     摘要：[目的]构建重组人骨形成蛋白7(rhBMP7)基因真核表达质粒，转染兔关节软骨细胞，探讨外源性人rhBMP7基因在转染软骨细胞中的表达情况及对细胞生物学性状的影响。[方法]采用PCR技术扩增rhBMP7基因，将其插入真核表达载体pcDNA 31中，体外分离培养兔关节软骨细胞，然后用构建的rhBMP7质粒转染软骨细胞，经G418筛选、免疫组化染色和逆转录PCR检测其表达，同时检测表达产物对维持软骨细胞表型的作用。[结果]经过PCR及酶切鉴定证实获得了BMP7真核表达质粒pcDNA 31rhBMP7，通过免疫组化染色和逆转录PCR鉴定证实rhBMP7在转染后的软骨细胞中得到了表达，其表达产物促进软骨细胞表型的维持。[结论]外源性rhBMP7基因转染软骨细胞可以获得高效表达，并具有一定的维持软骨细胞表型的作用，为软骨组织工程的研究提供了改良的种子细胞。

    关键词：骨形成蛋白7；基因转染；软骨细胞

    Construction of eucaryotic expression plasmid carrying the recombined human BMP7 gene and its expression in chondrocytes∥LU Huading,CAI Daozhang,MA Huihui,et alDepartment of Orthopaedics,the 3rd Affiliated Hospital,Sun YatUniversity,Guangzhou 510630

    Abstract：[Objective]To construct an eucaryotic expression plasmid carrying the recombined human BMP7 gene,and transfect into rabbits articular chondrocytes in vitro,then study its expression in chondrocytes,and make further study on the effects of transfection on biolotical characteristics of these cells[Method]The rhBMP7 gene was cloned into the eucaryotic expression vector pcDNA31At the same time,rabbits articular chondrocytes were isolated and cultured in vitroThe plasmid carrying the rhBMP7 gene was transfected into chondrocytesThen the expression of the transgene was detected by using RTPCR assay and immunohistochemical examination after G418 riddling[Result]PCR and digesting demonstrated that the eucaryotic expression plasmid pcDNA31rhBMP7 was obtainedRTPCR and immunohistochemical examination showed that the rhBMP7 gene was expressed in chondrocytes,and the transgene expression products promote proliferation of chondrocytes[Conclusion]The exogenous rhBMP7 gene could be expressed by transfected chondrocytes effectively,and the transgene expression products could promote maintaining the phenotype of chondrocytesIt provides an ideal seeds for cartilage tissue engineering ......

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