重组质粒pEGFP_BMP7的构建及在大鼠骨髓间充质干细胞中的表达
Construction of Recombinant Plasmid pEGFP-BMP7 and Its Expression in Rat Bone Marrow Mesenchymal Stem Cellsin vitro
作者：胡 静,戚孟春,邹淑娟,李继华,周海孝
Author：HUJing1,QIMeng-chun2,ZOUShu-juan3,LIJi-hua1,ZHOUHai-xiao1
收稿日期： 年卷（期）页码：2005,23(06):463-
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：骨形成蛋白7,基因克隆,基因转染,间充质干细胞,
Key words：bone morphogenetic protein 7,gene clone,cell transfection,mesenchymal stem cells,
基金项目：
国家自然科学基金资助项目(30371553)
中文摘要
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。
英文摘要
Objective To construct recombinant plasmid pEGFP-BMP7 and determine its expression in rat bone marrow mes- enchymal stem cells (MSCs)in vitro.Methods cDNA of target gene was obtained from neonatal rat kidney by RT-PCR. After sequencing the target gene, the cDNAwas 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 deter- mined. RT-PCR and immunocytochemical analysiswere also performed to detectthe expression of BMP7 in ratMSCs.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 cDNAwas 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 marrowMSCs. This procedure may provide a unique method for stimulation of callus formation in distraction osteogenesis and reconstruction of craniofacial bone defects.
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