CAJ | 학술논문

目的构建双表达骨形态发生蛋白（BMP）9、6重组腺病毒并探讨其对C3H10细胞成骨的影响。方法以单一表达的BMP9或BMP6 AdEasy质粒为模板，PCR自扩增BMP9和BMP6基因序列，先后将其插入穿梭质粒pASG2，获得双表达穿梭质粒pASG2-BMP9、6，然后与腺病毒骨架质粒pAdEasy-1在BJ5183中同源重组，酶切鉴定成功后，转染至HEK293细胞中包装和扩增得到高滴度双表达腺病毒AdBMP9、6，体外转染C3H10细胞，RT-PCR方法检测BMP9、BMP6 mRNA水平的表达，早期成骨指标碱性磷酸酶染色、晚期指标钙茜素红染色检测其成骨作用。结果成功构建双表达重组腺病毒AdBMP9、6，RT-PCR证实双表达腺病毒能成功转染C3H10细胞，并且转染的C3H10细胞早晚期成骨指标碱性磷酸酶染色及钙茜素红染色活性较单一表达BMP9或BMP6的腺病毒组均增强。结论双表达重组腺病毒载体AdBMP9、6具有定向诱导C3H10细胞成骨分化的能力，且较单一表达BMP9或BMP6强。
목적구건쌍표체골형태발생단백（BMP）9、6중조선병독병탐토기대C3H10세포성골적영향。방법이단일표체적BMP9혹BMP6 AdEasy질립위모판，PCR자확증BMP9화BMP6기인서렬，선후장기삽입천사질립pASG2，획득쌍표체천사질립pASG2-BMP9、6，연후여선병독골가질립pAdEasy-1재BJ5183중동원중조，매절감정성공후，전염지HEK293세포중포장화확증득도고적도쌍표체선병독AdBMP9、6，체외전염C3H10세포，RT-PCR방법검측BMP9、BMP6 mRNA수평적표체，조기성골지표감성린산매염색、만기지표개천소홍염색검측기성골작용。결과성공구건쌍표체중조선병독AdBMP9、6，RT-PCR증실쌍표체선병독능성공전염C3H10세포，병차전염적C3H10세포조만기성골지표감성린산매염색급개천소홍염색활성교단일표체BMP9혹BMP6적선병독조균증강。결론쌍표체중조선병독재체AdBMP9、6구유정향유도C3H10세포성골분화적능력，차교단일표체BMP9혹BMP6강。
Objective To construct a recombinant adenovirus co-expressing bone morphogenic protein (BMP) 9 and BMP6 and observe its effect on the osteogenesis in C3H10 cells. Method The full-length sequences of BMP9 and BMP6 were amplified from AdEasy vector by PCR and cloned into the shuttle plasmid pASG2 vector to construct the co-expression shuttle plasmid pASG2-BMP9, 6 followed by homologous recombination with plasmid pAdeasy-1 in BJ5183. After confirmation by restriction endonuclease digestion, the recombinant vector was transfected into HEK293 cells, and high-titer recombinant adenovirus (Ad-BMP9, 6) was collected after amplification. Ad-BMP9, 6 was then transduced into C3H10 cells in vitro, and the mRNA expression of BMP9 and BMP6 was detected by RT-PCR. The osteogenic capability of the transfected cells was observed by alkaline phosphatase staining and calcium-alizarin red staining. Results AdBMP9,6 was constructed successfully and effectively infected in C3H10 cells, in which high expressions of BMP6 and BMP9 were detected. C3H10 cells infected with Ad-BMP9,6 showed stronger alkaline phosphatase and calcium-alizarin red staining than the cells trasnfected by either BMP9 or BMP6 alone. Conclusion The recombinant adenovirus co-expressing BMP9 and BMP6 we constructed shows a more potent effect than the adenoviruses expressing either BMP9 or BMP6 alone in inducing the osteogenic differentiation of C3H10 cells into osteoblasts.