Ultrasound-Mediated Microbubble Destruction Enhances Bone Morphogenetic Protein-2 Gene Expression

The ideal result of periodontal therapy is not only to end pathological changes themselves, but also to promote periodontal regeneration, thereby to achieve the complete recovery of the periodontal tissue physiologically and functionally. Bone Morphogenetic Proteins is kindred of cell cytokines which could highly induce osteogenesis. Among all the hypotypes, BMP-2 is a kind of strong bone-inducing factor and plays a crucial role in periodontal regeneration. But relying on the exogenous implantation only, the effective time and local concentration of BMPs are too limited to repair the bone injury, and to fulfill the goal of periodontal regeneration. Now, gene therapy technique has been introduced into the field of periodontology as a new hope to reach the target.There are ways to import the target gene transfer in vitro and in vivo. The former refers to transfecting the target gene into the target cell in vitro so as to form genetically modified cell expressing foreign gene, then planting the genetically modified cell back into vivo in order to make it play biological roles. Accordingly, gene transfer in vitro is considered as complicated due to the need of cell culture and cell Sorting. The latter is tansfering the gene of interest into the target cell directly in vivo. The transferred gene in the target cell would transcript and express so as to play a therapeutic effect. It does not require cell culture and transplant, this method is simple, economic, security, as well as closer to the actual clinical application. Yet, the greatest difficulty in using in vivo transfer method currently is the low transfection efficiency of target gene.At present, there are mainly two kinds of gene vectors:viral vectors and non viral vectors. Viral vectors are constructed based on certain viral structure and transfection mechanism, which has the advantage of high transfection efficiency. but the complex preparation,as well as its high immunogenicity and non-directive characters greatly impose restrictions to the clinical application of viral delivery system. Non viral vectors are primarily refer to plasmid vectors,the use of which has the disadvantage of low transfection efficiency. So it need to enhance the transfection efficiency combining with physical or chemical methods. Chemical method mainly uses lipids, peptides, polymers and other chemical substances, such substances are with a positive charge, through the role of charge-coupled it can carry DNA with negative charge into cells. However, this technology also has shortcomings of non-specificity in gene transfection site and low transfection efficiency in vivo.With regard to physical methods, Electroporation is currently more in-depth research and application.It refers to the use of high-intensity electron field led to a temporary membrane pore opening, thus promoting exogenous DNA into the interior of the cell. This method is an effective method in vitro, but the required high-intensity current could make obvious damage to the tissue, which limits its application in living tissue. Therefore, the development of gene therapy needs to develop an efficient and safe gene transfer vectors.Direct injection of naked plasmid is recognized as an easy and effective way, and with high security. It is considered the direction of development of gene therapy.Recently,scholars at home and abroad found that ultrasound microbubble can be used as a novel gene transfection vector in vivo.Under certain ultrasonic irradiation, in addition to promoting diretional transfer of the target gene safely and effectively, it could augment the conversion rate and the expression level of exogenous genes. Accordingly, this method may become a new breakthrough in gene therapy research.The viral and non-viral delivers all have merits and demerits, so to develop a novel and effective method to deliver gene becomes a new investigation aim of the gene therapy research. Recently, some studies demonstrated that ultrasound-mediated microbubble destruction could enhance the transfection efficiency and expression of the exogenous gene to a certain orientation safely and effectively. This method would become a new breakthrough of the gene therapy.Therefore, the author planned to construct recombinant eukaryotic expression plasmid pIRES-rhBMP2-EGFP containing the enhanced green fluorescence protein (EGFP) and the recombinant human bone morphogenetic protein-2 (rhBMP-2) by PCR, T/A cloning; in addtion, the author also investigates the transfection efficiency of rhBMP-2 gene in targeted cells by ultrasound-mediated microbubble destruction. The recombinant plasmid pIRES-rhBMP2-EGFP containing the enhanced green fluorescence protein (EGFP) and the recombinant human bone morphogenetic protein-2 (rhBMP-2) was transferred into mouse skeletal muscle using Ultrasound-Mediated Microbubble Destruction method. Then we detected the transfection efficiency of rhBMP-2 gene in vivo targeted cell using ultrasound-mediated microbubble destruction to evaluate the feasibility of this method in periodontal tissue regeneration.Part 1:Construction of a Eukaryotic Expression Vector Containing the Enhanced Green Fluorescence Protein and the Recombinant Human Bone Morphogenetic Protein-2Objective:To construct the eukaryotic co-expression plasmid which contains EGFP and rhBMP-2 gene using plasmid pIRES as the vector by way of recombinant DNA technology in vitro.Methods:1. A pair of primers specific for amplifying the DNA fragment encoding rhBMP-2 were designed and synthesized. The targeted DNA fragment was amplified from pIRES-rhBMP2 by PCR.2. rhBMP-2 and EGFP were inserted to the proper sites of vector pIRES, and internal ribozyme entry site (IRES) sequence was between the genes coding for rhBMP-2 and EGFP.3. The recombinant plasmid pIRES-rhBMP2-EGFP was first propagated in E.coli DH5α, and then was confirmed to contain hBMP2cDNA sequence by agarose gel electrophoresis and DNA sequence analysis. Results:The construction of the recombinant eukaryotic dual-expression plasmid pIRES-rhBMP2-EGFP and the correct of the open reading frame were confirmed through restriction enzyme maping analysis and DNA sequencing.Conclusion:By PCR,T/A cloning, the cDNA fragment encoding rhBMP2 and EGFP fragment can be cloned into pIRES to construct the recombinant eukaryotic expression plasmid pIRES-rhBMP2-EGFP.Part 2:Ultrasound-Mediated Microbubble Destruction Enhances Exogenous Gene Expression in NIH3T3 Cells in VitroObjective:Using mice NIH3T3 as vector cell, to explore whether ultrasound-mediated microbubble destruction can enhance the efficiency of gene transfection in vitro cells.It provides a practicability for appling ultrasound-mediated microbubble destruction to the regeneration of tissue in vivo.Methods:1. NIH3T3 cells were anabiosised and gone down to the 3rd to 4th generation, then cultured into 6 well plates.2. The cells were divided into 2 groups:plasmid DNA+ LipofectamineTM 2000 group(D+LF); plasmid DNA+ultrasound+microbubble group(D+U+M), and then plasmid DNA was transfected into cells with liposome or ultrasound and microbubble.3.24～48 hours later, EGFP was applied to observe the expression of plasmid by fluoresence microscope, and the concentrations of BMP-2 were evaluated by enzyme-linked immunosorbent assay (ELISA).4. The results were analyzed by cure fitting and t-test of SPSS 11.5.Results:The transfection efficiency rate is 7.30±1.58% in D+LF group, but 11.77±3.16% in D+U+M group(P<0.05); The concentration of BMP-2 after transfection is 1164.35±724.67pg/ml in D+LF group, but 2932.70±656.27 pg/ml in D+U+M group(P<0.05).Conclusion:Ultrasound-mediated microbubble destruction could enhance the transfection efficiency and expression of rhBMP-2 gene in NIH3T3 cells. This provides a new safe and effective gene delivery system for gene therapy in periodontal regeneration.Part 3:Ultrasound-Mediated Microbubble Destruction Enhances Bone Morphogenetic Protein-2 Gene Expression in mouse skeletal muscleObjective:The recombinant BMP-2 palsmid pIRES-rhBMP2-EGFP containing enhanced green fluorescence protein was transfected into mice skeletal muscle cells in vivo using ultrasound-mediated microbubble destruction technology.to explore whether micobubbles could enhance the expression of BMP-2 gene in mice.lt would help for making certain the practicability of gene therapy with the aid of ultrasound-mediated microbubble destruction and providing the theory basis for the application of direct gene therapy method mediated by above technology in the periodontal tissue regeneration.Methods:Twenty four male BALB/c mice were divided into four groups. The naked plasmid was injected into the pretibial muscle or the quadriceps muscle (group A and group C) without Ultrasound-Mediated Microbubble Destruction method. Micobubb- les with plasmid were injected into the pretibial muscle or the quadriceps muscle (group B and group D) with destructing microbubbles by ultrasound immediately. Twelve mice (group A and group B,30μg plasmid injected) were killed after 7 days and the tissue samples of the pretibial muscle were obtained to observe the expression of EGFP by Inverted fluorescence microscope, gene transfection efficiencies were quantified by counting EGFP positive fibers on mice skeletal muscle. After 14 days, the other twelve mice (group C and group D, 100μg plasmid injected) were killed and immunnohistochemical technique was applied to detect the rhBMP-2 gene expression.Results:The percentage of EGFP-positive fibers was much lower in the group A than that in the group B. The difference between the two groups was significant. After 14 days, BMP-2 was defected in the group C and group D, but expression efficiency of BMP-2 in the group D was much higher.Conclusion:Ultrasound-mediated microbubble destruction could enhance the transfection and expression efficiency of rhBMP-2 gene in skeletal muscle of mouse in vivo. These results suggest that ultrasound mediated microbubble destruction method may become a possible new strategy in direct gene injection therapy of bone morphogenetic protein-2 for periodontitis.