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《Chongqing Medicine》 2010-09
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Construction of eukaryotic expression vector pEGFP-N_1-BMP_2 and its expression in human periodontal ligament fibroblast cells transfected via ultrasound-mediated microbubble destruction

ZHONG Lin,ZHONG Xiao-bo,ZHANG Yun-yan,et al.(Department of Endodontics,Affiliated Hospital of Stomatology,Chongqing Medical University,Chongqing 400015,China)  
Objective To construct the recombinant eukaryote plasmid pEGFP-N1-BMP2 and to detect its transient expression in HPDLFs transfected by ultrasound-mediated microbubble destruction.Methods Total RNA was extracted from human placenta trophoblastic cells.The hBMP2 cDNA was obtained by RT-PCR and inserted into pMD19-T plasmid.After sequencing the recombinant plasmid pMD19-T-BMP2,the hBMP2 cDNA was inserted into the pEGFP-N1 vector,and then the pEGFP-N1-BMP2 vector was identified by double digestion and transfected into HPDLFs by ultrasound-mediated microbubble destruction.Its expression was detected by fluorescent microscopy and RT-PCR.Results The hBMP2 gene fragment was successfully inserted into pEGFP-N1 and confirmed by restriction endonuclease analysis and DNA sequencing.After the transfection of pEGFP-N1-BMP2 into HPDLFs(cultured in vitro)by ultrasound-mediated microbubble destruction,it could be expressed transiently in HPDLFs,which could be confirmed by fluorescent microscopy and RT-PCR.Conclusion Recombinant eukaryotic expression vector pEGFP-N1-BMP2 can be successfully constructed and transfected into HPDLFs by ultrasound-mediated microbubbles destruction;and the hBMP2 gene can be expressed successfully in HPDLFs.This basis provides a foundation for further studies in the applications of pEGFP-N1-BMP2 and ultrasound microbubble on gene therapy for periodontal regeneration.
【CateGory Index】: R78
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