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目的:利用基因工程技术构建人骨形态发生蛋白2重组腺病毒,并进行体内表达,测定活性;为深入开展的基因治疗研究创造条件。方法:实验于2000-05/2002-06在西安交通大学第一附属医院骨科实验室完成。应用PCR技术扩增出人骨形态发生蛋白2全长基因,体外同源重组构建重组腺病毒后,随机将30只SD大鼠分为治疗组和对照组,每组15只,将纯化后的重组腺病毒50μL注入SD大鼠股部肌肉陷窝病损处,通过组织学染色、X射线片对动物模型的组织变化进行观测。结果:6周后治疗组可见明显骨诱导形成,2周时治疗组碱性磷酸酶活性明显高于对照组,重组腺病毒治疗组有明显的诱导成骨活性。结论:应用于动物实验中目的基因表达,并具有生物学活性。本研究是在骨缺损基因治疗的一次尝试,且人骨形态发生蛋白2重组腺病毒的构建成功,也为国内骨科疾病基因治疗的进一步研究提供了基础。
OBJECTIVE: To construct recombinant human bone morphogenetic protein-2 recombinant adenoviruses by genetic engineering and to express in vivo and assay their activity. Methods: The experiment was performed at the Department of Orthopedics, the First Affiliated Hospital of Xi’an Jiaotong University from May 2000 to June 2002. PCR amplification of human bone morphogenetic protein 2 full-length gene, in vitro homologous recombination to construct recombinant adenovirus, 30 SD rats were randomly divided into treatment group and control group, 15 in each group, the purified recombinant Fifty microliters of adenovirus was injected into the lesion of the muscle in the femoral part of SD rats, and the histological changes of the animal model were observed by X-ray. Results: After 6 weeks, the bone formation was observed in the treatment group. At 2 weeks, the alkaline phosphatase activity in the treatment group was significantly higher than that in the control group. The recombinant adenovirus treatment group had obvious osteogenic activity. Conclusion: It is used in animal experiments for gene expression and has biological activity. This study is an attempt to gene therapy of bone defects, and the successful construction of human bone morphogenetic protein 2 recombinant adenovirus also provided the basis for further research on the gene therapy of orthopedic diseases in China.