摘要
目的:构建骨形态发生蛋白4(BMP4)重组腺病毒,探讨其对NIH3T3成纤维细胞向成骨方向分化的影响。方法:将BMP4基因克隆连接到载体pAdTrack-CMV中,在细菌BJ5183中与pAdEasy腺病毒基因组进行同源重组,得到BMP4重组腺病毒基因组,通过转染HEK293细胞包装出重组腺病毒。利用BMP4重组腺病毒转染NIH3T3细胞,采用逆转录-多聚酶链反应(RT-PCR)和western-blot检测BMP4在细胞中的mRNA和蛋白表达。Gomori改良钙钴法检测BMP4重组腺病毒转染后的NIH3T3细胞碱性磷酸酶表达;von kossa染色检测BMP4重组腺病毒转染后NIH3T3细胞的成骨分化情况。结果:获得了BMP4转移质粒pAdTrack-BMP4和BMP4重组腺病毒基因组,并包装出重组腺病毒。BMP4在转染后的NIH3T3细胞中得到表达,表达的BMP4蛋白具有生物学活性。转染后的NIH3T3细胞碱性磷酸酶表达增加。BMP4促进了NIH3T3细胞向成骨方向分化,形成钙结节。结论:本实验成功构建了BMP4重组腺病毒;BMP4重组腺病毒可促进NIH3T3细胞向成骨方向分化。
Objective To construct a recombinant adenovirus carrying BMP4 gene which is transfected into NIH3T3 cells and expressed. Methods BMP4 gene was cloned into the shuttle vector pAdTrack-CMV; the bacterium BJ5183 was contransfected with the shuttle and the pAdEasy vector and the recombinant adenoviral plasmid was produced by homologous. Recombinant adenovirus was packaged in HEK293 cells. NIH3T3 cells were transfected with recombinant adenovirus and BMP4 expression was confirmed by reverse transcription-polymerase chain reaction（RT-PCR）and western blot. Modified Gomori method was performed on 3th and 6th day after transfection with BMP4 adenovirus, yon kossa staining was used to evaluate the osteoblast differentiation. Results PCR and digesting demonstrated that the shuttle plasmid pAdTrack-BMP4 and the recombinant adenoviral plasmid were obtained. The recombinant adenovirus was packaged in HEK293 cells. The results of RT-PCR and western blot revealed that BMP4 was expressed in NIH3T3 cells. ALP expression was increased significantly in the NIH3T3 cells transfected with BMP4 adenovirus, yon kossa staining confirmed the osteoblast differentiation in the cells transfected with BMP4 adenovirus. Conclusion Construction of BMP4 recombinant adenovirus and its expression in NIH3T3 laid the basis for BMP4 gene therapy for bone repair.
出处
《中国运动医学杂志》
CAS
CSCD
北大核心
2008年第3期294-297,共4页
Chinese Journal of Sports Medicine
基金
国家体育总局科研项目基金
教育部博士点基金(20060001140)共同资助
