转BMP-7基因BMSCs复合nHAC体外培养的实验研究

目的建立能够稳定表达骨形态发生蛋白-7（BMP-7）的骨髓基质干细胞（BMSCs）,并与纳米羟基磷灰石胶原（nHAC）材料复合培养体外构建组织工程骨。方法用慢病毒把人BMP-7基因和增强型绿色荧光蛋白（eGFP）基因导入大鼠BMSCs,用G418筛选获得阳性细胞后接种于nHAC支架体外复合培养。荧光显微镜下观察eGFP表达,判断感染效率;以四唑盐（MTT）实验检测细胞活力;RT-PCR、ELISA检测目的基因表达;碱性磷酸酶（ALP）活性检测成骨能力;扫描电镜观察细胞在支架材料中的黏附、生长状况。结果慢病毒24h对BMSCs的转染率约为90%;MTT实验显示细胞活性较未转染组无明显差异（P〉0.05）;RT-PCR检测到BMP-7表达,在1300bp处出现特异性条带;ELISA检测24hBMP-7蛋白含量为（150.2±18.3）pg/ml;ALP活性以第16天左右最强;扫描电镜见细胞种植nHAC支架后粘附、生长良好。结论 BMP-7可在BMSCs内稳定表达,并诱导其向成骨分化;与nHAC复合培养可构建组织工程骨。

In vitro co-culture of BMSCs transfected with lentivirus-mediated BMP-7 gene with nHAC scaffold

Objective To establish modified bone marrow stromal cells （BMSCs） which can express BMP-7 stably; then BMSCs were planted into the scaffold of nano-hydroxyapatite/collagen （nHAC） material to construct tissue engineering bone. Methods BMP-7 and eGFP genes were transfected into BMSCs of SD rats by using lentiviruses. The positive clonies were selected by using G418 and co-cultured in vitro with nHAC scaffold. The transfection efficiency and proliferation of BMSCs were measured under the fluorescence microscopy and MTT method, respectively. The expression of BMP-7 was detected by RT-PCR and ELISA. Osteogenous differentiation was tested with ALP activity. The adhesion and growth of BMSCs on the nHAC scaffold were observed under the scanning electron microscopy （SEM）. Results The transfection efficiency was about 90% in the first 24 h. There was no significant difference in proliferations between transfected and non-transfected BMSCs （P〉0.05）. RT-PCR revealed that BMP-7 gene was located at 1300 bp in transfected group but not in the other groups. ELISA confirmed the BMP-7 expression was （150.2±18.3） pg/ml. The ALP activity was strongest nearly on the 16th day. The adhesion and growth were very well on the nHAC scaffold. Conclusion BMP-7 gene was stably expressed in BMSCs, and could enhance the capacity of osteogenous differentiation. The BMSCs modified by BMP-7 combined with nHAC can construct tissue engineering bone.