腺病毒介导的骨形态发生蛋白-2和骨形态发生蛋白-7基因联合转染大鼠脂肪来源干细胞对成骨分化的影响

Effects of adenovirus-mediated co-transfection of bone morphogenetic protein-2 gene and bone mor- phogenetic protein-7 gene on osteogenic differentiation of rat adipose-derived mesenchymal stromalcells

目的观察腺病毒介导的骨形态发生蛋白一2（BMP-2）和骨形态发生蛋白-7（BMP．7）基因联合转染对大鼠脂肪来源干细胞成骨分化的影响。方法分别构建携带绿色荧光蛋白的BMP-2重组腺病毒表达质粒及携带红色荧光蛋白的BMP-7的重组腺病毒表达质粒，包装、生产携带BMP-2和BMP-7的腺病毒。分离并培养大鼠脂肪来源干细胞（ADSCs），分别以Ad．BMP-2转染ADSCs、Ad—BMP一7转染ADSCs和Ad—BMP-2、Ad—BMP-7共转染ADSCs，另设未转染ADSCs作为对照。转染后在荧光显微镜下观察4组细胞中重组蛋白的表达。应用Westernblot检测转染后14d4组细胞骨钙素（OCN）和I型胶原的蛋白表达，并检测各组碱性磷酸酶（ALP）的活性。结果荧光显微镜下观察BMP-2和BMP-7可以高效稳定表达，骨钙素（OCN）和I型胶原的表达明显升高，差异有统计学意义（P〈0．05），并且联合转染组最高。Ad—BMP-2及Ad—BMP-7转染细胞的ALP活性升高，联合转染组的ALP活性明显高于其他组，差异有统计学意义（P〈0．01）。结论腺病毒介导BMP-2和BMP一7基因共转染可促进ADSCs向成骨方向分化。

Objective To investigate the effects of adenovirus-mediated co-transfection of bone morphogenetic protein-2 （BMP-2） gene and bone morphogenetic protein-7 （BMP-7） gene on osteogenic differentiation of adipose-derived mesenchymal stromal cells （ADSCs）. Methods The expression adenovi- rus vectors with green fluorescent protein gene carrying BMP-2 and adenovirus vectors with red fluorescent protein gene carrying BMP-7 were packaged and produced respectively. Rat adipose-derived stromal ceils were isolated and cultured in vitro, and were transfected with Ad-BMP-2 （ Ad-BMP-2 group） , Ad-BMP-7 （Ad-BMP-7 group）, co-transfected with Ad-BMP-2 and Ad-BMP-7 （Ad-BMP-2 ＋ Ad-BMP-7 group）, or transfected with no virus （control group）. The expression of osteocalcin （OCN） and collagen I protein at 14th day after transfection was detected by using Western blotting in each group. Alkaline phosphatase （ALP） activity in ADSCs was measured. Results The expression of OCN and collagen I protein in ~b- transfected group was significantly higher than that in the other groups （P 〈 0.05 ）.The ALP activity in co- transfected group was significantly stronger than that in the other groups （P 〈 0. O1 ）. Conclusion Adeno- virus-mediated co-transfection of BMP-2 gene and BMP-7 gene may promote osteogenic differentiation of ad- ipose-derived ADSCs.