作　　者： ; ; ; ; ;

机构地区： 四川大学华西口腔医学院

出　　处： 《华西口腔医学杂志》 2005年第6期463-466,共4页

摘　　要： 目的体外构建重组质粒pEGFP_BMP7,并检测其在大鼠骨髓间充质干细胞中的表达。方法应用RT_PCR方法从大鼠肾脏中分离、扩增目的基因片段,并进行测序,随后将所得cDNA定向克隆到真核表达载体pEGFP_N1中,进行双酶切来鉴定克隆的正确性。通过脂质体介导重组pEGFP_BMP7瞬时转染大鼠骨髓间充质干细胞,确定转染效率,并通过RT_PCR、免疫细胞化学手段检测BMP7的表达。结果通过RT_PCR成功获得1.3 kb的cDNA片段,该cDNA除756 bp处有一碱基从T突变成A外,其余序列与大鼠BMP7基因完全相符。重组质粒双酶切图谱显示,BMP7 cDNA被正确插入载体中。绿色荧光蛋白在大鼠骨髓间充质干细胞中早期瞬时转染效率可达33%。转染后RT_PCR和免疫细胞化学检测证实重组pEGFP_BMP7在大鼠骨髓间充质干细胞中的表达。结论成功构建重组真核表达质粒pEGFP_BMP7,并在大鼠骨髓间充质干细胞中得到表达,有助于应用BMP7行基因治疗,促进牵张成骨、骨痂形成和修复颅颌面骨缺损。 Objective To construct recombinant plasmid pEGFP-BMP7 and determine its expression in rat bone marrow mesenchymal stem ceils （MSCs） in vitro.Methods cDNA of target gene was obtained from neonatal rat kidney by RT-PCR. After sequencing the target gene, the cDNA was subcloned into a eukaryote plasmid pEGFP-N1 by directed cloning and then digested with two restrictive endonucleases to verify the correctiveness of the recombinant plasmid pEGFP-BMP7. Rat bone marrow MSCs were transiently transfected with the pEGFP-BMP7 and transfection efficiency of the Green Fluorescent Protein （GFP） was determined. RT-PCR and immunocytochemical analysis were also performed to detect the expression of BMP7 in rat MSCs. Results A 1 311 bp cDNA fragment was obtained by RT-PCR and sequence analysis showed it matched perfectly with that of rat BMP7 gene except a single nucleotide change at 756 bp from T to A. Digestion of the recombinant plasmid showed two 1.3 kb and 4.7 kb fragments and their size were same as those of BMP7 and pEGFP, This indicated that BMP7 cDNA was successfully subcloned into pEGFP. Transient transfection showed an efficiency of 33 % at day 2 in rat MSCs. After transfection, transcription of BMP7 was detected in MSCs and expression of BMP7 protein was also verified. Conclusion Recombinant eukaryote plasmid pEGFP-BMP7 was successfully constructed and expressed in rat bone marrow MSCs. This procedure may provide a unique method for stimulation of callus formation in distraction osteogenesis and reconstruction of craniofacial bone defects.

关 键 词： 骨形成蛋白 基因克隆 基因转染 间充质干细胞

领　　域： [生物学]