论文标题:腺病毒介导的人骨形成蛋白2（Ad-hBMP2）基因转染促进兔下颌牵张成骨（DO）的实验研究
Bone Morphogenetic Proteins2 Gene Therapy for Mandibular Distraction Osteogenesis in Rabbits
论文作者 陈安威
论文导师 魏奉才,论文学位 硕士,论文专业 口腔临床医学
论文单位 山东大学,点击次数 71,论文页数 63页File Size8263k
2005-05-06论文网 http://www.lw23.com/lunwen_200926917/ 重组腺病毒;骨形成蛋白2;牵张成骨;下颌骨;基因治疗
recombinant adenovirus ; Bone Morphogenetic Protein 2; distraction osteogenesis; mandible; gene therapy
目的: 1.骨形成蛋白2(Bone Morphogenetic Protein 2,BMP2)是转化生长因子β(Transforming Growth Factor β,TGF β)超家族的成员之一,也是生长因子的一种。它的主要生物学作用是诱导未分化的间充质细胞分化增殖,形成软骨和新生骨。所以具有巨大的潜在临床应用价值。本实验将建立扩增、纯化和生物活性鉴定腺病毒介导的人BMP2(Ad-hBMP2)重组腺病毒的有效方法,为进一步研究应用Ad-hBMP2进行兔下颌牵张成骨基因治疗奠定基础。 2.牵张成骨(distraction osteogenesis,DO)是近年发展起来的一项颇有价值的外科技术,它具有极大的临床应用潜力。虽然牵张成骨成功率很高,但却需要一个很长的固定阶段并且确有失败发生。骨形成蛋白类具有促进骨折和阶段性骨缺损愈合的作用,但它们对下颌骨牵张成骨的作用仍存初步研究中。本实验通过在兔下颌骨牵张成骨模型上局部应用Ad-hBMP2,探讨其在DO过程中的作用和机理,以寻找一种缩短DO疗程的新方法。 方法: 实验一,应用人胚肾293细胞作为包装细胞,扩增携带hBMP2表达片段的重组腺病毒载体Ad-hBMP2和携带增强型绿色荧光蛋白(enhance green fluorescent protein,EGFP)表达片段的重组腺病毒载体Ad-EGFP,利用氯化铯密度梯度超速离心法对其进行纯化,应用50%感染量测定(infectious dose 50,ID_(50))方法对纯化病毒液进行病毒滴度测定。选用6只成年新西兰大白兔(体重3.0～3.5kg),分别在两侧股部肌肉以100μl的
Objective:1. Bone Morphogenetic Protein 2 (BMP2) is one of members of Transforming Growth Factor (3 (TGF p) superfamily and one of growth factors. The major biological role of BMP2 is to induce undifferentiated mesenchymal cell into cartilage and newly formed bone. There is enormous potential value in clinical application. We shall raise a modus operandi to amplificate and purify recombinant adenovirus vectors containing BMP2 gene and to accredit it"s biological activity. That is foundation to bone morphogenetic proteins 2 gene therapy for mandibular distraction osteogenesis in rabbits.2. Distraction Osteogenesis (DO) is a valuable clinical technology developed in recent years, which have a great potential in clinical applications and gave us a new way to study the basic mechanisms of bone regeneration. It requires a long consolidation period and has a low but real failure rate. Bone Morphogenetic Proteins have effectiveness on promoting fracture union and stage bone coloboma union. But the study of their effectiveness on mandibular distraction osteogenesis is initial. In this study, we were to applicate ad-hBMP2 topically on the basis of the established DO animal model in rabbits and to detect the action in bone regeneration of DO, in order that we might find a new way to improve bone regeneration of DO.Methods:1.The cell 293 ,a human embryonic kidney cell line as a packaging cellwas used for culture of recombinant adenovirus vectors containing hBMP2 gene and recombinant adenovirus vectors containing EGFP gene, adenovirus vectors were reproduced and purified after 2 rounds of cscl centrification, biological activity identification was carried out using infectious dose 50. 6 adult male New Zealand rabbits weighting between 3.0-3.5 kilograms were used in this study, the bilateral Muscle of thigh were injected respectively with lOOul ad-hBMP2 and Ad-EGFP. Other treatment was identical. Three rabbits were killed randomly at the 14th and 28th day after operation. All the specimens were X-rayed and histologically examinated.2. 36 adult male New Zealand rabbits weighting between 3.0-3.5 kilograms were used in this study. Under general anesthesia, an incision on the skin along the inferior margin of mandible was performed. The soft tissue was separated and the surface of bone was exposed. Then mandibular osteotomy was done on the edentulous area about 1 mm before the first premolar. In operation, periosteum, bone marrow and the inferior alveolar artery should be carefully protected. After good hemostasia, the cut bone was fixed with a self-prepared bilateral extra-oral bone-supported distractor. Then periosteum was sutured and the wound was closed seriously. The bone stumps were fixed in situ for 7 days after operation(latency period), then distractors were activated at a speed of 0.5 millimeter every 12 hours for 7 days before they were fixed(consolidation period). At The 15th day after operation one side of all the animals"distraction gaps were injected lOOul ad-hBMP2, 18 sides of other sides were injected lOOul ad-EGFP and 18sides received no treatment. 12 rabbits including 12 Ad-BMP2-treated sides, 6 Ad-EGFP control sides and 6 blank sides were killed randomly at the 7th day, the 14th and 28th day of consolidation period separately. Specimens were observed, and inspections were taken with X-ray, histomorphology and immunohistochemical techniques. Results:I. We have succeed to culture cell 293, recombinant adenovirus vectors containing hBMP2 and purify adenovirus. The purified recombinant adenovirus vectors containing hBMP2 was obtained titer of 1.58xlOnpfu/ml in this study. Skeletogenous cells and cartilage cells were observed in the sides of Ad-hBMP2 treatment and there were not density change of the two sides two weeks after operation. Four weeks after operation new bone had formed, trabeculae of bone, cavitas medullaris bone marrow and small amounts lymphocytes around of circum- muscle were observed, there were obviously density change in the sides of Ad-hBMP2 tre
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