Ultrasound-Mediated Microbubble Destruction Enhances Human Bone Morphogenetic Protein-2 Gene Expression In Human Periodontal Ligament Fibroblast Cells

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。...