Liu Xiaoyun, Li Kanghua, Song Jianhua, Liang Changyong, Wang Xiyang, Chen Xinwen
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, People's Republic of China.
Spine (Phila Pa 1976). 2006 Apr 1;31(7):732-5. doi: 10.1097/01.brs.0000206977.61305.43.
A recombinant baculovirus vector expressing GFP (Ac-CMV-GFP) was tested in rabbit intervertebral disc cells cultured in monolayer in vitro. Direct infection of intervertebral disc cells was then assessed in vivo.
To test the efficacy of the baculovirus-mediated marked gene transfer to rabbit intervertebral disc cells, and the expression of the transgene in vitro and in vivo.
The potential application of gene therapy for the treatment of degenerative disc disease has been an area of exciting and active research. Many efforts have been made toward the construction of delivery vectors using mammalian viruses, such as adenovirus, adeno-associated virus, retrovirus, and herpesvirus. However, recently, baculovirus-derived vectors have emerged as possible tools for gene transfer into mammalian cells in vitro and in vivo but, to our knowledge, have never been tested in an intervertebral disc cell.
The intervertebral disc cells cultured in monolayer were treated with 6 different doses of baculovirus carrying the green fluorescence protein gene (Ac-CMV-GFP). Fluorescence microscopy and flow cytometry were used to analyze transgene expression. The Autographa californica nucleopolyhedrovirus/GFP virus was then injected directly into the intervertebral discs of 8 rabbits at 7, 13, 20, and 28 days after injection. The nucleus pulposus tissues of injected discs were evaluated immediately by fluorescence microscopy for GFP expression.
A dose of Ac-CMV-GFP at a multiplicity of infection of 200 achieved the highest transduction ratio (approximately 87% of nucleus pulposus cells) and long-term expression without any toxicity to the cells. In vivo assay showed that Ac-CMV-GFP could also mediate GFP expression in rabbit intervertebral disc cells without inducing any symptoms. The GFP expression level at 7 days after transduction was significantly higher than at 21 and 28 days after treatment.
Baculovirus can transfer exogenous genes into rabbit nucleus pulposus cells at a high efficiency, and safely both in vitro and in vivo. The results suggest that baculoviruses might be useful tools as gene therapy vectors for intervertebral disc diseases.
在体外单层培养的兔椎间盘细胞中测试表达绿色荧光蛋白的重组杆状病毒载体（Ac-CMV-GFP）。然后在体内评估椎间盘细胞的直接感染情况。
测试杆状病毒介导的标记基因转移至兔椎间盘细胞的效果，以及转基因在体外和体内的表达情况。
基因治疗在退行性椎间盘疾病治疗中的潜在应用一直是一个令人兴奋且活跃的研究领域。人们已做出许多努力来构建使用哺乳动物病毒的递送载体，如腺病毒、腺相关病毒、逆转录病毒和疱疹病毒。然而，最近，杆状病毒衍生的载体已成为体外和体内将基因转移至哺乳动物细胞的可能工具，但据我们所知，从未在椎间盘细胞中进行过测试。
用6种不同剂量携带绿色荧光蛋白基因的杆状病毒（Ac-CMV-GFP）处理单层培养的椎间盘细胞。使用荧光显微镜和流式细胞术分析转基因表达。然后在注射后第7、13、20和28天，将苜蓿银纹夜蛾核型多角体病毒/绿色荧光蛋白病毒直接注射到8只兔的椎间盘中。注射椎间盘后立即通过荧光显微镜评估髓核组织中的绿色荧光蛋白表达。
感染复数为200的Ac-CMV-GFP剂量实现了最高的转导率（约87%的髓核细胞）和长期表达，且对细胞无任何毒性。体内试验表明，Ac-CMV-GFP也可介导兔椎间盘细胞中的绿色荧光蛋白表达，且不引发任何症状。转导后第7天的绿色荧光蛋白表达水平显著高于处理后第21天和第28天。
杆状病毒可在体外和体内高效、安全地将外源基因转移至兔髓核细胞。结果表明，杆状病毒可能是用于椎间盘疾病基因治疗载体的有用工具。
