Nishida K, Kang J D, Suh J K, Robbins P D, Evans C H, Gilbertson L G
Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pennsylvania, USA.
Spine (Phila Pa 1976). 1998 Nov 15;23(22):2437-42; discussion 2443. doi: 10.1097/00007632-199811150-00016.
In vitro and in vivo studies using a rabbit model were performed to determine the feasibility of adenovirus-mediated gene transfer to the intervertebral disc.
This study was conducted to determine whether it is possible to transfer genes to cells within the intervertebral disc by direct injection of an adenovirus and to determine the duration of gene expression obtained by this method.
Although growth factors have the potential to stimulate the regeneration of nucleus pulposus, sustained delivery of growth factors to a degenerated disc is clinically unfeasible with present technology. Novel approaches such as gene transfer should be investigated as possible solutions to this problem.
The lacZ marker gene was used to evaluate gene delivery to cells within intervertebral discs. For the in vitro study, cell cultures were established from the nucleus pulposus tissue of New Zealand white rabbits and infected with an adenovirus encoding the lacZ gene (Ad-lacZ). For the in vivo study, the anterior aspects of lumbar intervertebral discs were surgically exposed, and Ad-lacZ in saline solution was directly injected into the nucleus pulposus. An equal volume of saline only was injected into control discs. Expression of the transferred gene was detected by staining with 5-bromo-4-chloro-3-indolyl-beta-galactosidase (X-Gal).
The in vitro experiments confirmed that nucleus pulposus cells were efficiently transduced by an adenoviral vector carrying the lacZ gene. In vivo injection of Ad-lacZ into the nucleus pulposus resulted in the transduction of a considerable number of cells. Marker gene expression in vivo persisted at an apparently undiminished level for at least 12 weeks. No staining was noted in control discs.
The results show the feasibility of adenovirus-mediated gene transfer to the intervertebral disc. Expression of the marker gene persisted at least 12 weeks in vivo. This successful demonstration of exogenous gene transfer to the disc and sustained, long-term expression suggests that the adenoviral vector may be suitable for delivery of appropriate genes to the disc for the treatment of spinal disorders.
采用兔模型进行体外和体内研究，以确定腺病毒介导的基因转移至椎间盘的可行性。
本研究旨在确定通过直接注射腺病毒将基因转移至椎间盘内细胞是否可行，并确定通过该方法获得的基因表达持续时间。
尽管生长因子有刺激髓核再生的潜力，但以目前的技术，将生长因子持续递送至退变椎间盘在临床上不可行。应研究诸如基因转移等新方法，作为解决该问题的可能方案。
使用lacZ标记基因评估基因向椎间盘内细胞的递送。体外研究中，从新西兰白兔的髓核组织建立细胞培养物，并用编码lacZ基因的腺病毒（Ad-lacZ）感染。体内研究中，手术暴露腰椎间盘的前部，将盐溶液中的Ad-lacZ直接注入髓核。对照椎间盘仅注射等体积的生理盐水。通过用5-溴-4-氯-3-吲哚基-β-半乳糖苷（X-Gal）染色检测转移基因的表达。
体外实验证实，携带lacZ基因的腺病毒载体可有效转导髓核细胞。向髓核内体内注射Ad-lacZ导致大量细胞被转导。体内标记基因表达至少持续12周，水平未见明显下降。对照椎间盘中未观察到染色。
结果表明腺病毒介导的基因转移至椎间盘具有可行性。标记基因在体内表达至少持续12周。外源基因成功转移至椎间盘并持续长期表达，表明腺病毒载体可能适用于向椎间盘递送合适基因以治疗脊柱疾病。
