【作者】 仲琳；

【导师】 钟晓波；

【作者基本信息】 重庆医科大学 ， 口腔临床医学， 2010， 硕士

【摘要】 近年来以生长因子局部应用为代表的牙周再生治疗受到广泛关注和研究。骨诱导形成蛋白-2(BMP2)是唯一能够单独诱导成骨的生长因子,在牙周组织中的分子调节作用也已经比较明确。它可诱导牙周组织中未分化间充质细胞分化为成骨细胞和成牙骨质细胞,对人牙周膜成纤维细胞(HPDLFs)具有成骨定向分化诱导作用,但局部直接使用时存在着易流失、效率低等问题。基因治疗技术的应用可以使外源性的BMP2直接在宿主细胞内完成表达和后加工,减少全身的毒副作用,增强了局部治疗效果,为牙周组织再生提供了广阔的研究前景。导入方法的选择是决定基因治疗效果的关键因素。超声微泡转基因技术作为一种新型的、安全有效的基因转染方法,目前已应用于心脏、血管、肝脏、肾脏、骨骼肌、眼等领域的基因转染实验研究中,已有大量研究表明,超声介导微泡造影剂破裂可增强目的基因在体内外的转染与表达。但该技术用于人牙周组织的报道较少,本课题以hBMP2为目的基因将超声微泡转基因技术用于转染HPDLFs,并观察转染后目的基因在HPDLFs中的表达情况,为该技术在牙周再生基因治疗方面的进一步研究奠定实验基础。目的:构建带有目的基因hBMP2的真核表达载体pEGFP-N1-BMP2,然后利用超声微泡转基因技术将其转入HPDLFs,并检测转染后目的基因的表达情况。方法:1.采用组织块法原代培养HPDLFs,通过形态学及免疫组织化学鉴定后,进行传代培养,取3～5代细胞用于实验。2.根据Genbank数据库中hBMP2的cDNA序列(Gen-Bank,Accession No.NM001200),利用Primer Premier 5设计软件设计并合成引物,从人胎盘滋养层细胞系中提取总RNA,采用RT-PCR方法获得hBMP2基因,连接pMD19-T载体后,转化大肠杆菌DH5a并筛选阳性克隆,经酶切鉴定及测序正确后,用EcoRⅠ和BamH I限制性内切酶分别双酶切pMD19-T-BMP2与pEGFP-N1质粒,再将回收、纯化后的BMP2基因和pEGFP-N1质粒进行连接、转化,筛选pEGFP-N1-BMP2的阳性克隆,鉴定正确后用于后续实验。3.利用超声微泡转基因技术,在声强为0.5 w/cm~2,间断波辐照时间60 s,微泡浓度为15%的条件下将重组质粒pEGFP-N1-BMP2转入HPDLFs中,同时以在相同条件下转染空质粒pEGFP-N1组及未转染组做对照,通过荧光显微镜,RT-PCR和免疫荧光技术分别检测转染后HPDLFs中BMP2的表达情况。结果:1.牙周膜成纤维细胞原代培养成功并传代。2.成功克隆人BMP2基因,构建了真核表达载体pEGFP-N1-BMP2。3.超声微泡转基因技术在适当的条件下可将携带目的基因BMP2的重组质粒pEGFP-N1-BMP2成功转入HPDLFs;经RT-PCR及免疫荧光检测显示,与对照组相比pEGFP-N1-BMP2转染组中BMP2在HPDLFs内的瞬时表达明显增强。结论:成功构建pEGFP-N1-BMP2真核表达载体,并通过超声微泡转基因技术转入HPDLFs,增强了hBMP2在HPDLFs中的表达,为进一步研究该载体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。更多还原

【Abstract】 In recent years, extensive concern and research have been focused on the treatments of periodontal regeneration represented with the local application of growth factor. BMP2 is the only growth factor which can induce bone formation alone, and its molecular regulatory effects on the periodontal tissues have been relatively clear. It can induce differentiation from the undifferentiated mesenchymal cells in the periodontal tissues to osteoblasts and cementoblasts cell, and also has the oriented differentiation induction from HPDLFs to osteoblasts.However, many problems exist when BMP2 is directly used locally, such as easy loss, low efficiency and so on. The application of gene therapy techniques can make BMP2 finish expressing and post-processing in the host cells directly; reduce systemic side effects; enhance the effects of local treatments, and provide a broad study prospects for periodontal tissue regeneration. The Selection of gene transfection methods is important to determine the efficacy of gene therapy. Ultrasound microbubble transgenic technology, as a new effective and secure method of gene transfection in vivo, has been applied to the Experimental Study of gene transfer in the heart, blood vessels, liver, kidney, skeletal muscle, eye and other fields. Now a large number of studies have shown that ultrasound-mediated microbubble destruction can enhance the transfection efficiency and expression of target genes in vitro and in vivo. But the reports about ultrasound microbubbles in periodontal tissue are very few. In this study, Ultrasound microbubble transgenic technology is used to transfect BMP2 gene into HPDLFs and the expression of BMP2 in HPDLFs was detected after transfection.This provides a basis for further studies in the gene therapy of periodontal regenerationObjective:To construct the eukaryotic expression vector pEGFP-N1-BMP2 with the target gene hBMP2, then transfer pEGFP-N1-BMP2 into HPDLFs by ultrasound microbubble transgenic technology, and detect the expression of hBMP2 in HPDLFs after transfection.Methods:1. Adopt tissue explants culture technique for the primary culture of HPDLFs,then the primary cells were passaged after morphological and immunohistochemical identification. Take 3~5generations of HPDLFs for subsequent experiments. 2. According the cDNA sequence of hBMP2 from Genbank database (Gen-Bank, Accession No.NM001200), design and synthesize the primers using Primer Premier 5.0.software. Total RNA was extracted from human placenta trophoblastic cells. The hBMP2 cDNA was obtained by RT-PCR and inserted into pMD19-T vector, then transformed into E.coli DH5a and screen out the positive clone. After identifying by restriction endonuclease analysis and DNA sequencing, pMD19-T-BMP2 and pEGFP-N1 plasmid were double digested with restriction endonuclease EcoR I and BamH I, then connected the reclaimed and purified hBMP2 and pEGFP-N1 plasmid; transformed the recombinant plasmid pEGFP-N1-BMP2 into E.coli DH5a; and screened out the positive clone. After identifying correctly, take recombinant plasmid pEGFP-N1-BMP2 for subsequent experiments.3. transfer recombinant plasmid pEGFP-N1-BMP2 into HPDLFs by Ultrasound microbubbles transgenic technology under the conditions of sound intensity 0.5w/cm~2 ,the concentration of microbubbles 15%, intermittent wave irradiation time 60 s. Simultaneously, under the same conditions, compared with the group transfected with pEGFP-N1 and non-transfected group as controls. After transfection, detect the expression of BMP2 in HPDLFs by fluorescence microscopy, RT-PCR and immunofluorescence techniques.Results:1. The primary periodontal ligament fibroblasts are cultured and passaged successfully2. The hBMP2 gene is cloned and the eukaryotic expression vector pEGFP-N1-BMP2 is constructed successfully3. Ultrasound microbubbles transgenic technology can transfer the eukaryotic expression vector pEGFP-N1-BMP2 which carrying hBMP2 gene into HPDLFs successfully in the appropriate conditions; the results of RT-PCR and immunofluorescence showed that: comparing with the control groups, the expression of BMP2 in pEGFP-N1-BMP2 transfection group was enhanced obviously.Conclusions:The eukaryotic expression vector pEGFP-N1-BMP2 was constructed and transfected into HPDLFs by ultrasound microbubbles transgenic technology successfully; and the expression of BMP2 in the HPDLFs was enhanced after transfection.This provides a foundation for further studies in the applications of pEGFP-N1-BMP2 and Ultrasound microbubble on gene therapy for periodontal regeneration。更多还原