【作者】 孙钦峰；

【导师】 杨丕山； 姜广水；

【作者基本信息】 山东大学 ， 口腔临床医学， 2010， 博士

【摘要】 牙周疾病治疗的理想效果不仅在于终止病变本身,更在于促进牙周组织的再生,从而达到牙周组织结构与功能的完全恢复。骨形成蛋白(bone morphogenetic proteins, BMPs)是一种具有高度骨诱导活性的细胞因子家族,在众多的亚型中,骨形成蛋白-2(BMP-2)是BMPs家族中诱骨活性最强的一种,也是能单独诱导成骨的因子,其在牙周组织再生和修复中具有重要的作用。但仅靠外源性植入,BMPs在骨损伤部位存在的时间及浓度是有限的,往往不能满足某些骨损伤修复的需要,尚不能达到牙周组织完全再生的目的。基因治疗技术引入牙周病治疗的研究领域,为这一目标的实现带来了新的希望。导入目的基因的途径有体外转移法和体内转移法。前者将目的基因在体外转染靶细胞,形成表达外源基因的基因修饰细胞,然后回植体内以发挥生物学效应,因此体外转移法因需细胞培养和筛选等而复杂；后者是将目的基因在体内直接转移到靶细胞,使其进入靶细胞并转录、表达以发挥治疗作用。它不需要细胞培养移植,方法简便、经济、安全,更接近临床应用实际。但是,体内转移法目前遇到的最大困难就是目的基因转染效率低下。目前基因载体主要有病毒性载体与非病毒性载体两类。病毒型载体是基于某些病毒自身结构和转染机制而构建的,具有转染效率高的优点,但其制备复杂,而且具有高免疫原性和非导向性,大大限制了病毒性转运系统的临床应用。非病毒性基因载体主要有质粒载体,其转染效率较低,需结合物理或化学方法来提高。化学法主要采用脂类、肽类、多聚体等化学物质,此类物质带有正电荷,通过电荷偶联作用可将携带负电荷的DNA导入细胞。但是这种技术同样具有基因转染部位的非特异性及活体内转染效率低的缺点。物理法目前研究应用较多的为电穿孔法,电穿孔法系指使用高强度的电子场导致暂时性的细胞膜孔开放,由此促使外源性的DNA进入细胞内部[。此法体外应用效果较好,但其所需的高强度电流对组织的明显损害,限制了其在活体组织的应用。因此,基因治疗的发展需开发一种高效安全的基因转运载体。裸质粒直接注射法是公认的一种既简便又有效的方法,并且安全性高,被认为是基因治疗的发展方向。但是质粒的转染效率较低,所以寻找提高质粒转染效率的方法是目前基因治疗研究的热点之一。最近,国内外学者研究发现超声微泡造影剂可作为一种新型的体内基因转染载体,在一定条件的超声照射下能促进目的基因安全而有效的定向转移,增加外源性基因转化率和表达水平,该方法有可能成为基因治疗研究中新的突破点。因此在本文中,我们拟通过PCR、T/A克隆等DNA重组技术,构建含增强型绿色荧光蛋白(enhanced green fluorescence protein, EGFP)的重组人骨形成蛋白-2(recombinant human BMP-2, rhBMP-2)基因真核表达质粒；通过体外实验,探讨超声介导微泡破裂法对目的基因转染靶细胞的影响；同时,将含增强型绿色荧光蛋白的重组BMP-2质粒pIRES-rhBMP2-EGFP进行小鼠后肢骨骼肌体内转染,研究微泡造影剂在超声作用下能否促进BMP-2基因在小鼠体内表达,为其介导的BMP2直接基因疗法在组织再生中应用的可行性提供初步实验数据。实验一含增强型绿色荧光蛋白的重组人骨形成蛋白-2基因真核表达载体的构建目的：采用DNA体外重组技术,以质粒pIRES为载体,构建一含有EGFP和rhBMP-2基因的真核双表达质粒。方法：1.PCR扩增rhBMP-2基因,并在两端添加合适的酶切位点。2.利用定向克隆技术将rhBMP-2基因和EGFP基因分别插入到载体pIRES的上下游多克隆位点中,两者通过IRES连接以防止两者融合表达。3.重组的pIRES-rhBMP2-EGFP在大肠杆菌DH5a (escherichia coli DH5α, E.coli DH5α)内扩增后,通过酶切电泳鉴定和DNA测序证明质粒构建成功。结果：通过对重组质粒pIRES-rhBMP2-EGFP进行酶切鉴定以及DNA序列测定分析,证明真核表达重组质粒pIRES-rhBMP2-EGFP构建成功,开放阅读框架正确。结论：利用PCR、T/A克隆等分子生物学技术可成功地将编码rhBMP-2的DNA片段和EGFP片段克隆到载体pIRES中,构建出重组子pIRES-rhBMP2-EGFP.实验二超声介导微泡破裂法促进骨形成蛋白-2基因在NIH3T3细胞中的表达目的：以小鼠NIH3T3为载体细胞,探讨超声介导微泡破裂法促进体外细胞基因转染效率,为超声微泡破裂法在体内组织再生中的应用提供可行性。方法：1.细胞培养：复苏NIH3T3细胞并传代3到4次,待细胞生长状态良好后接种至六孔培养板。2.质粒DNA的转染：六孔板中细胞随机分为两组,分别采用质粒DNA+脂质体法(D+LF组)和质粒DNA+超声+微泡法(D+U+M组)转染目的基因。3.检测项目与观察指标：转染24-48h后对各组细胞分别进行荧光显微镜下计数以计算转染效率,并进行酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)以测定转染后hBMP-2蛋白浓度。4.使用SPSS 11.5软件包分析实验结果：采用t检验对两组转染效率进行分析,采用曲线拟合和t检验对ELISA结果进行分析,P<0.05表示有统计学意义。结果：脂质体组的转染效率为7.30±1.58%,超声介导微泡破裂组为11.77±3.16%(P<0.05);脂质体组的转染后hBMP-2蛋白浓度为1164.35±724.67pg/ml,超声介导微泡破裂组为2932.70±656.27 pg/ml。超声介导微泡破裂组对转染率和hBMP-2蛋白浓度均显著高于脂质体组(P<0.05)。结论：超声介导微泡破裂法可以明显提高外源性基因rhBMP-2在体外NIH3T3细胞中的转染效率和蛋白浓度,可以为牙周再生基因治疗提供一种新型基因转移系统。实验三超声微泡破裂法促进骨形成蛋白-2在小鼠骨骼肌中的表达目的：采用超声微泡破裂法将含增强型绿色荧光蛋白的重组BMP-2质粒pIRES-rhBMP2-EGFP进行小鼠后肢骨骼肌体内转染,研究微泡造影剂在超声作用下能否促进BMP-2基因在小鼠体内表达,有助于确定借助超声微泡破裂法进行BMP2基因治疗的可行性,并为其介导的BMP2直接基因疗法在牙周组织再生中的应用提供初步理论依据。方法：1、24只BALB/c小鼠随机分为4组,每组6只：A组：在小鼠右侧胫前肌注射注入质粒与生理盐水的混合溶液(含质粒30μg);B组：在小鼠右侧胫前肌注入质粒与超声微泡造影剂混合溶液(含质粒30μg)后立即用超声辐照;C组：在小鼠右侧股四头肌注入质粒与生理盐水的混合溶液(含质粒100μg);D组：在小鼠右侧股四头肌注入质粒与超声微泡造影剂混合溶液(含质粒100μg)后立即用超声辐照。2、7天后处死A组和B组取小鼠胫前肌观察绿色荧光蛋白的表达情况。14天后处死C组和D组取小鼠股四头肌免疫组化检测rhBMP2表达情况。结果：7天后B组绿色荧光阳性肌纤维百分率高于A组；14天后,C组和D组都可检测到rhBMP2的表达,但D组BMP-2表达量多于C组。结论：超声介导微泡破裂法能增加外源性rhBMP-2基因在小鼠体内骨骼肌转化率和表达水平。这些结果显示超声介导微泡破裂法为牙周BMP2直接法基因治疗提供一种新型方法。更多还原

【Abstract】 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.更多还原