双表达骨形态发生蛋白9、7重组腺病毒载体的构建及其对间充质干细胞C3H10的成骨作用

Construction of Recombinant Adenovirus Co-expressing BMP9,BMP7 and Effect on Osteogenesis of Mesenchymal Stem Cells C3H10

目的构建双表达骨形态发生蛋白(BMP)9、7腺病毒重组体并进行初步成骨鉴定。方法自单一表达的BMP9或BMP7 AdEasy质粒上扩增BMP9和BMP7基因序列,先后定向亚克隆至同一穿梭质粒pASG2,获得双表达穿梭质pASG2-BMP9、7。酶切、PCR鉴定及测序正确后与骨架质粒pAdEasy-1同源重组获得双表达BMP9、BMP7腺病毒质粒,转染至HEK293细胞中包装和扩增得到高滴度双表达BMP9、BMP7腺病毒,体外转染C3H10细胞,碱性磷酸酶染色及钙茜素红染色检测其早晚期成骨作用。结果成功构建双表达BMP9、BMP7的腺病毒,RT-PCR证实双表达腺病毒在C3H10细胞中表达,其转染的C3H10细胞早期碱性磷酸酶染色及晚期钙茜素红染色活性较单一表达的BMP7或BMP9腺病毒组增强。结论成功构建双表达BMP9、BMP7的重组腺病毒载体,其重组体具有定向诱导C3H10细胞成骨分化的能力。

Objective To construct a recombinant adenovims co-expressing BMP9 and BMP7, and identify its primary effect on osteogen- esis. Methods The gene sequence of BMP9 and BMP7 were amplified from AdEasy vector by PCR and sub-cloned into pASG2 vector. The co-expression shuttle plasmid pASG2-BMP9,7 was confirmed by restriction endonuclease digestion,PCR and gene sequencing;then plasmid pAdeasy-1 was co-transfected to produce recombinant adenovirus plasmid by homologous recombination. In addition,the recombinant vector was lransfected into HEK293 cells. High-titer recombinant adenovirus （Ad-BMP9, 7） was collected after rounds of amplification. The capability of osteogenesis induced with Ad-BMP9, 7 was evaluated in vitro in C3H10 cells by alkaline phosphatase staining and calcium- alizarin red staining. Results Ad-BMP9, 7 was constructed successfully. Testing with C3H10 cells ,alkaline phosphatase staining and calci- um-alizarin red staining were stronger than single transfected BMUI or BMP9 cells. Conclusion The recombinant adenovirus co-expressing BMP9 and BMP7 was constructed successfully,which has a capability of inducing osteogenic differentiation of C3H10 cells.