构建过表达hBMP-7基因重组腺病毒及体外转染犬骨髓基质细胞的研究

Construction of hBMP-7 Gene Recombinant Adenovirus and Its Expression after Infecting dMSCs

目的:构建携带人骨形态发生蛋白7(hBMP-7)基因的重组腺病毒载体,体外转染犬骨髓基质干细胞(dMSCs),检测hBMP-7的表达。方法:利用AdEasy腺病毒表达系统,体外构建高效表达hBMP-7重组腺病毒载体,酶切电泳鉴定后体外转染dMSCs,检测转染情况及目的基因的表达。结果:酶切鉴定表明pAd-hBMP-7已成功构建,体外可高效转染dMSCs,RT-PCR方法及免疫细胞化学检测表明hBMP-7可在dMSCs中表达。结论:成功克隆hBMP-7基因并构建重组腺病毒表达载体,证实了其在dMSCs中的表达,为火器伤致颌骨缺损的早期局部基因治疗打下基础。

Objective： To study the effect of recombinant pAd-human bone morphogenetic protein 7（pAd-hBMP-7） on osteogenic differentiation of dog marrow stromal cells（dMSCs）.Methods： Recombinant pAd-hBMP-7 was constructed and the titer of recombinant adenovirus was determined.pAd-hBMP-7 and pAdTrack-CMV were used to transfect dMSCs.Transfection efficiency was measured by fluorescent microscope.hBMP-7 expression was detected by RT-PCR and immunocytochemical analysis. Results： The titer of pAd-hBMP-7 reached about 1.25 ×l0^10 PFU/rnl and transfection efficiency of exogenous gene was nearly 80 after 48h. After transfection, transcription of hBMP -7 and expres- sion of hBMP - 7 protein were also verified in dMSCs by RT - PCR and immunocytochemical analysis. Condusion： The resuits of this study indicated that recombinant pAd - hBMP - 7 can successfully transfect dMSCs and accelerate their osteogenic differentiation. The technique explored in this study provides a unique and valuable gene engineering approach for reconstruction of craniofaeial bone defects.