重组35型腺病毒介导人骨形成蛋白基因对脂肪源性干细胞的诱导分化

DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS INDUCED BY RECOMBINANT ADENOVIRUS'S CONTAINING FIBERS DERIVED FROM B-GROUP SEROTYPE 35-MEDIATED BONE MORPHOGENETIC PROTAIN

目的在体外运用人骨形成蛋白7(human bone morphogenetic protein7,hBMP-7)基因重组35型腺病毒(recombinant adenovirus's containing fibers derived from B-group serotype35,rAd5/F35)诱导大鼠脂肪源性干细胞(adipose-derived adult stem cell,ADASC)向成骨细胞定向分化,为骨组织工程探索新的细胞来源。方法以pcDNA1.1/氨苄青霉素-hBMP-7质粒为模板扩增hBMP-7基因,将回收的PCR产物片段克隆入pDC316载体,获得重组质粒pDC316-hBMP-7。骨架质粒pBHG-fiber5/35和穿梭质粒pDC316-hBMP-7共转染293细胞,同源重组产生rAd5/F35-hBMP-7。同样的方法构建rAd5/F35-增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)。在体外分别转染大鼠ADASC并留置空白对照。观察细胞形态和生长情况,ELISA检测转染基因的转录和表达,检测钙结节和骨钙素等成骨细胞表型。结果PCR、酶切鉴定表明rAd5/F35-hBMP-7质粒构建正确。rAd5/F35-EGFP对大鼠ADASC中转染效率可达90%以上,hBMP-7基因存在于转染后的ADASC中并表达相应的蛋白,诱导组钙结节形成,电镜见胞质中有钙质成分,碱性磷酸酶阳性,骨钙素表达增强。结论rAd5/F35介导hBMP-7基因可促进ADASC向成骨细胞定向分化。

Objective To investigate the differentiation of the adipose-derived adult stem cell （ADASC） induced by the recombinant adenovirus＇s containing fibers derived from B-group serotype 35 （rAd5/F35）-mediated human bone morphogenetic protein 7 （hBMP-7） gene and to explore a new cell source for the bone tissue engineering. Methods The hBMP-7 gene was amplified with the pcDNA1. 1/AMP hBMP-7 plasmid as a formwork. After the purification, the gene fragment was cloned into the pDC316 carrier for the recombination of the plasmid of pDC316- hBMP-7. The 293 cells were co transfected by the skeleton plasmid of pBHG-fiber5/35 and the shuttle plasmid of pDC316-hBMP-7, and the recombinant plasmid of Ad5/F35-hBMP-7 was obtained; the recombinant plasmid of Ad5/ F35-enhancd green fluorescent protein（EGFP） was obtained by the similar method. The rat ADASCs were cultured and transfected by the Ad5/F35-hBMP-7 plasmid and the Ad5/F35-EGFP plasmid, respectively; the remaining untransfected ADASC were used as the controls. The morphology and the growth pattern of the transfected cells were evaluated. The transcription and the expression of the transfected genes and the steogenic phenotypes such as calcium nodules and osteocalcin were evaluated by ELISA. Results The identification of PCR and enzyme cutting showed that the construction of the recombinant Ad5/F35-hBMP-7 plasmid could be confirmed. The transfection rate of the ADASC by the AdS/F35-EGFP plasmid was determined to be greater than 90%. The hBMP-7 gene in the transfected ADASC could express the corresponding protein, and the formation of the calcium nodules could be found in the induced group. The electron microscopy showed that there was a calcium element in the cytoplasm, the alkaline phosphatase result was positive, and the expression of osteocalcin was increased. Conclusion The rAd5/F35 hBMP-7 gene can promote the differentiation of the adipose-derived adult stem cells to the osteoblasts in the bone tissue engineering.