【作者】 王娟；

【导师】 孙卫斌；

【作者基本信息】 南京医科大学 ， 口腔临床医学， 2006， 硕士

【摘要】 牙周治疗的理想目标就在于诱导牙周组织再生。新生牙槽骨是重建牙周新附着的基础，没有新生的牙槽骨，牙周纤维组织就没有附着的锚地。骨形成蛋白(bone morphogenetic protein，BMP)属于转化生长因子β超家族成员，能在非骨组织如肌肉中诱导新骨形成，因而在骨生长和骨缺损治疗中极为重要。已有大量动物实验研究结果表明：局部运用外源性重组BMP可以诱导牙周损伤区域牙槽骨、牙骨质、牙周纤维组织的新生。但是，牙周组织的特点决定了局部应用外源性BMP既很难达到所要求的浓度，又极易被体液稀释流失，而且在一定程度上还有促进细菌繁殖的可能。为了克服外源性生长因子半衰期短、生物活性低、易降解的缺点，现多将基因工程与牙周组织工程相结合，通过转基因手段诱导表达分泌型生长因子，在局部造成生长因子适当表达并持续一段时间，为利用生长因子诱导牙周组织再生开拓了新的前景。本研究拟将人骨形成蛋白2真核表达载体pcDNA3.1-B2转染入小鼠成纤维细胞系细胞(NIH3T3细胞)，建立能稳定表达BMP2的细胞系，并利用细胞共培养系统Transwell，观察转基因诱导表达的分泌型BMP2对NIH3T3细胞骨化分化的影响。以探讨BMP2基因治疗在牙周组织工程方面的意义。 实验一 pcDNA3.1-B2真核表达质粒的鉴定及稳定表达BMP2细胞系的建立 目的 通过转基因技术，建立稳定表达BMP2的成纤维细胞系。 材料和方法 将pcDNA3.1-B2用EcoR Ⅰ和Xba Ⅰ双酶切后，经1％琼脂糖凝胶电泳鉴定，并检测其携带目的基因的核酸序列。将经过鉴定的pcDNA3.1-B2真核表达质粒通过高效的阳离子聚合物转染试剂转染入NIH3T3细胞中，并用G418筛选获得稳定转染细胞株。RT-PCR和酶联免更多还原

【Abstract】 The reconstruction of lost periodontal tissue including bone, ligament, and cementum is a major goal of therapy. Bone regeneration is the beginning of periodontal tissue regeneration, which can induce periodontal ligament fiber’s attachment. Bone morphogenetic protein (BMP), which belongs to the transforming growth factor [beta] superfamily, BMP are powerful regulators of cartilage and bone formation during embryonic development and regeneration in post-natal life. Some studies have indicated that exogenous rhBMP could promote the proliferation and DNA synthesis of the human periodontal ligament cells and induce mesenchymal precursor cells in the human periodontal ligament to differentiate into osteoblasts and cementoblasts. In order to overcome the disadvantage of exogenous BMP, for example of short half time, poor biological activity and rapid diffusion, gene engineering now frequently was integrated with periodontal tissue engineering. The growth factor which is induced by gene transfection can locally express suitably and maintain relative long period. This way will be helpful in the induction of periodontal regeneration. The aim of this study is to establish fibroblasts that stably expressing human bone morphogenetic protein 2 by transfecting eukaryotic expression vector (pcDNA3.1-B2) into NIH3T3 cells. Then the effect of the secretive BMP2 induced by gene transfection on the ultrastructure, alkaline phosphatase (ALP) activity and osteocalcin (OCN) expression of NIH3T3 cells were observed through Transwell system.更多还原