摘要
目的:研究表明,外源性重组骨形态发生蛋白7具有促进椎间盘细胞增殖以及促进细胞外基质合成的能力,为探讨骨形态发生蛋白7基因疗法治疗椎间盘退变的可行性,本实验拟构建重组人骨形态发生蛋白7的腺病毒载体,并观察其在原代培养的兔椎间盘髓核细胞中的表达情况。方法:实验于2005-12/2006-06在长海医院胸心外科研究所完成。从整合有人骨形态发生蛋白7全长cDNA的真核表达质粒pcDNA3.1(+)-骨形态发生蛋白7中聚合酶链反应扩增骨形态发生蛋白7基因的开放读码框,通过穿梭质粒整合到腺病毒载体pAd-Easy系统上,在293细胞中包装成熟、扩增,酶切鉴定人骨形态发生蛋白7基因整合到该载体中;该载体转染原代培养的兔髓核细胞,并采用反转录-聚合酶链反应和蛋白免疫印迹的方法检测其表达情况。结果:①实验成功建立了安全、稳定、有效的复制缺陷重组腺病毒Ad-骨形态发生蛋白7的构建方法,经鉴定后证实有骨形态发生蛋白7全长cDNA整合,并且为正向插入。②体外培养的兔原代髓核细胞表现出代谢活性低、增殖能力弱等特点;髓核细胞能够被腺病毒载体高效感染并能够高效表达不同的目的基因,感染效率随感染倍数值的增加而增加;当感染倍数=100时,95%以上的髓核细胞可以被感染,且细胞毒性较小,为最佳感染倍数。③采用最佳感染倍数转染髓核细胞后骨形态发生蛋白7基因在转染后3d就开始表达,并可以持续表达3周以上,几乎所有髓核细胞都可以表达骨形态发生蛋白7。结论:腺病毒载体可以作为进行骨形态发生蛋白7基因治疗椎间盘退变研究的有效载体。
AIM： Some research have confirmed that the exogenous recombinant bone morphogenetic protein-7 （BMP-7） can promote the intervertebral disc cell proliferation, as well as enhance the synthesis of extracellular matrix. The present study aimed to explore the feasibility of gene therapy with BMP-7 for intervertebral disc degeneration, to construct recombinant adenovirus vector carrying human bone morphogenetic protein-7 （hBMP-7） gene and to observe its expression in primary cultured rabbit nucleus pulposus cells.
METHODS： Experiments were performed at the Institute of Cardiothoracic Surgery of Changhai Hospital from December 2005 to June 2006. The open reading frame of hBMP7 was amplified from hBMP-7 full length cDNA that was carried in an eukaryotic plasmid pcDNA3.1 （＋）-BMP7 by polymerase chain reaction （PCR）, then subcloned into a shuttle plasmid integration, and the resultant plasmid was co-transduced with pAdEasyl plasmid to construct a new plasmid pAd-BMP7. The plasmid was transfected into 293 cells. The virus infected rabbit nucleus pulposus cells and the expression was detected in different levels by reverse transcriptase （RT）-PCR and Western-blot.
RESULTS： Replication defect Ad-BMP7 was constructed successfully. BMP7 full-length cDNA integration, norientation was detected. In vitro primary cultured nucleus pulposus cells had the characteristics of low metabolism rate and low ability of proliferation. After infected by adenoviral vector, the nucleus pulposus cells could express the gene. The efficacy of infection increased with the increase in multiplicity of infection （MOI）. When MOI was equal to 100, over 95% of nucleus pulposus cells were infected. The adenoviral had low toxicity. It was the MOI of choice. The expression began three days after BMP-7 gene transfection and it sustained over 3 weeks. BMP-7 could be expressed in almost all the nucleus pulposus cells.
CONCLUSION： Adenovirus vector is an ideal vector for BMP-7 gene therapy on researching intervertebral disc degeneration.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第28期5477-5481,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
作者简介
石健，男，1972年生，黑龙江省绥化市人，汉族，2006年解放军第二军医大学毕业，博士，主治医师，主要从事脊柱退变，脊髓损伤方面的研究。shjianzlk@hotmail．com
