pEGFP-N_1-BMP_2真核表达载体的构建及其经超声微泡介导转染HPDLFs后的表达  

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

作　　者：仲琳[1] 钟晓波[1] 张云燕[1] 骆书美[1] Nameeta Shrestha 齐进[1] 李春光[1] 

机构地区：[1]重庆医科大学附属口腔医院牙体牙髓科,400016

出　　处：《重庆医学》2010年第9期1045-1047,1050,F0003,共5页Chongqing medicine

摘　　要：目的构建pEGFP-N1-BMP2真核表达质粒,并检测其经超声微泡转基因技术转染人牙周膜成纤维细胞(HPDLFs)后的瞬时表达情况。方法从人胎盘滋养层细胞系中提取总RNA,采用RT-PCR方法获得目的基因人骨形成蛋白-2(hBMP2)基因片段,连接pMD19-T载体并测序正确后与真核荧光表达载体pEGFP-N1连接,酶切鉴定后利用超声微泡转基因技术转染入HPDLFs中,通过荧光显微镜和RT-PCR检测目的基因在HPDLFs中的表达。结果成功克隆人BMP2基因,重组质粒pEGFP-N1-BMP2经PCR及双酶切鉴定均证实hBMP2基因已与pEGFP-N1正确重组。pEGFP-N1-BMP2经超声微泡转染HPDLFs后,通过绿色荧光观察和RT-PCR检测证实hBMP2能够在体外培养的HPDLFs内有效的转录和瞬时表达。结论成功构建pEG-FP-N1-BMP2真核表达载体,通过超声微泡转基因技术成功转染入HPDLFs并得到有效表达,为进一步研究该质粒与超声微泡转基因技术在牙周再生基因治疗中的应用提供实验基础。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.

关 键 词：牙周再生 超声微泡 人骨形成蛋白-2 增强型绿色荧光蛋白 基因转染 人牙周膜成纤维细胞 

分 类 号：R78[医药卫生—口腔医学]