人骨形态发生蛋白－7腺病毒的构建及其在犬转染骨髓基质干细胞后的表达

摘要

Objective To explore the construction method of the recombinant adenovirus carrying human bone mor -phogenetic protein-7(BMP-7) and its expression after transfection of canine bone marrow stromal stem cells .Methods To construct vectors that carrying human bone morphogenetic protein -7 adenovirus in vitro using the adenoviral expression system and transfect into the human embryonic kidney 293 cells for homologous recombination, using recombinant adenovirus carrying human BMP-7 gene to transfect canine bone marrow mesenchymal stem cells , then to detect BMP-7 gene expression by the RT-PCR method.Results Restriction analysis proved that the BMP -7 transfer plasmid shuttle vector pTrack -BMP-7 and BMP-7 recombinant adenovirus genome had been obtained and a recombinant adenovirus was packaged , the virus titer was 3.6 ×1012 VP/ml.RT-PCR proved that BMP-7 gene was expressed in canine bone marrow mesenchymal stem cells which were transfected by recombinant adenovirus , the transfection efficiency was about 98%.Conclusion The successful construction of BMP-7 recombinant adenovirus and the successful transfection of canine bone marrow mesenchymal stem cells and their high expressions provide an experimental basis for the gene treatment of bone defect and nonunion .%目的：探讨携带人骨形态发生蛋白－7（BMP－7）腺病毒构建方法以及其在犬转染骨髓基质干细胞中的表达情况。方法采用腺病毒表达系统体外构建携带 BMP－7腺病毒的载体，转染人胚肾293细胞进行同源重组，利用 BMP－7重组腺病毒转染犬骨髓基质干细胞，然后采用 RT－PCR 法检测 BMP－7基因的表达。结果经过酶切鉴定证明获得了 BMP－7转移质粒穿梭载体 pTrack－BMP－7和 BMP－7重组腺病毒基因组，并包装出重组腺病毒，病毒滴度为3．6×1012 VP／ml。 RT－PCR 证明 BMP－7在重组腺病毒转染后的犬骨髓间充质干细胞中得到了表达，转染效率约98％。结论 BMP－7重组腺病毒的成功构建与成功转染犬骨髓间充质干细胞以及其高效表达，为骨缺损与骨不连的基因治疗提供了实验依据。