Moon S H, Gilbertson L G, Nishida K, Knaub M, Muzzonigro T, Robbins P D, Evans C H, Kang J D
Musculoskeletal Research Center, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
Spine (Phila Pa 1976). 2000 Oct 15;25(20):2573-9. doi: 10.1097/00007632-200010150-00006.
Human intervertebral disc cells were cultured in monolayer and treated with adenovirus-containing marker genes to determine the susceptibility of the cells to adenovirus-mediated gene transfer.
To test the efficacy of the adenovirus-mediated gene transfer technique for transferring exogenous genes to human intervertebral disc cells in vitro.
Upregulated proteoglycan synthesis after direct in vivo adenovirus-mediated transfer of growth factor genes to the rabbit intervertebral disc has previously been reported. Before contemplating extending this approach to the treatment of human disc disease, it is necessary to demonstrate that human intervertebral disc cells are indeed susceptible to adenovirus-mediated gene transduction.
Human intervertebral disc cells were isolated from disc tissue obtained from 15 patients during surgical disc procedures. The cells were cultured in monolayer and treated with saline containing five different doses of adenovirus carrying the lacZ gene (Ad/CMV-lacZ), saline containing adenovirus carrying the luciferase gene (Ad/CMV-luciferase), or saline alone. Transgene expression was analyzed by 5-bromo-4-chloro-3-indolyl-beta-galactosidase (X-Gal) staining and luciferase assay.
Adenovirus efficiently transferred lacZ and luciferase marker genes to cells from degenerated discs as well as to cells from nondegenerated discs. A minimum dose of 150 MOI Ad/CMV-lacZ was found to be sufficient to achieve transduction of approximately 100% of disc cells-regardless of patient age, sex, surgical indication, disc level, and degeneration grade. No statistically significant difference in the luciferase activities could be detected in disc cell cultures from degenerated and nondegenerated discs treated with Ad/CMV-luciferase.
In vitro transducibility of human intervertebral disc cells by adenovirus is relatively insensitive to disc degeneration grade. Because the rate-limiting step for successful gene therapy is the ability to transfer genes efficiently to the target tissue, the achievement of efficient gene transfer to human intervertebral disc cells(using a direct, adenovirus-mediated approach) is an important and necessary step in the development of gene therapy strategies for the management of human intervertebral disc disorders.
将人椎间盘细胞进行单层培养，并用携带标记基因的腺病毒进行处理，以确定细胞对腺病毒介导的基因转移的敏感性。
测试腺病毒介导的基因转移技术在体外将外源基因转移至人椎间盘细胞的有效性。
先前有报道称，将生长因子基因直接在体内通过腺病毒介导转移至兔椎间盘后，蛋白聚糖合成上调。在考虑将这种方法扩展至人类椎间盘疾病治疗之前，有必要证明人椎间盘细胞确实易受腺病毒介导的基因转导影响。
从15例患者手术椎间盘操作中获取的椎间盘组织中分离出人椎间盘细胞。将细胞进行单层培养，并用含有五种不同剂量携带lacZ基因的腺病毒（Ad/CMV-lacZ）的盐水、含有携带荧光素酶基因的腺病毒（Ad/CMV-荧光素酶）的盐水或仅用盐水处理。通过5-溴-4-氯-3-吲哚-β-半乳糖苷（X-Gal）染色和荧光素酶测定分析转基因表达。
腺病毒能有效地将lacZ和荧光素酶标记基因转移至退变椎间盘的细胞以及非退变椎间盘的细胞。发现最低剂量为150 MOI的Ad/CMV-lacZ足以实现约100%的椎间盘细胞转导，而与患者年龄、性别、手术指征、椎间盘节段和退变程度无关。在用Ad/CMV-荧光素酶处理的退变和非退变椎间盘的细胞培养物中，未检测到荧光素酶活性有统计学显著差异。
腺病毒对人椎间盘细胞的体外转导能力相对不依赖于椎间盘退变程度。由于成功基因治疗的限速步骤是将基因有效转移至靶组织的能力，因此（采用直接的、腺病毒介导的方法）实现对人椎间盘细胞的有效基因转移是开发用于治疗人类椎间盘疾病的基因治疗策略的重要且必要步骤。
