Therapeutic effects of adenovirus-mediated growth and differentiation factor-5 in a mice disc degeneration model induced by annulus needle puncture.
Therapeutic effects of adenovirus-mediated growth and differentiation factor-5 in a mice disc degeneration model induced by annulus needle puncture.
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DOI：
10.1016/j.spinee.2009.10.006
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发表时间：
2010-01
期刊：
影响因子：
4.5
通讯作者：
Li, Xudong Joshua
中科院分区：
文献类型：
--
作者：
Liang, Haixiang;Ma, Shen-Ying;Feng, Gang;Shen, Francis H.;Li, Xudong Joshua
迄今为止用于治疗椎间盘退变（IDD）的治疗策略集中于缓解症状，而退变的逆转仍然是有效治疗IDD的重要挑战。生长和分化因子-5（GDF 5）的缺乏会导致早期椎间盘退变，具有增加椎间盘细胞增殖和细胞外基质蛋白表达的潜力。建立小鼠腰椎间盘退变模型，观察腺病毒GDF 5基因治疗的效果。比较不同大小针刺椎间盘的退变变化，建立小鼠腰椎间盘退变模型。评价携带GDF 5基因的腺病毒载体对小鼠椎间盘退变模型的体内基因治疗效果。通过针刺Balb/c小鼠的椎间盘建立腰椎间盘退变模型。之后，对椎间盘退变的基因治疗进行了评估。随机选择两只小鼠腰椎间盘，用30号针头穿刺，然后注射腺病毒，腺病毒已被改造为表达荧光素酶基因（Ad-Luc）或GDF 5基因（Ad-GDF 5）。通过生物发光成像、放射学和MRI扫描分析动物，然后在术后1、2、4或8周处死动物，并进行组织学和生化测定。经MRI T2加权扫描及组织学检查，所有椎间盘经不同大小的穿刺针穿刺后均出现退变。而30 G针损伤组椎间盘退变的发展较其他组更为可靠和温和。通过生物发光成像检测荧光素酶活性显示，腺病毒在注射后存活，并且导入的基因在6周内表达。在Ad-Luc或Ad-GDF 5注射的小鼠中，直到手术后4周都没有T2加权MRI信号。在第6周和第8周，Ad-GDF 5组可检测到T2加权信号，而Ad-Luc对照组无信号。在两组中，椎间盘高度指数百分比（%DHI）在损伤后1周显著降低（~ 20%），表明椎间盘退变的发展。第2周，Ad-GDF 5组小鼠的DHI %较Ad-Luc组显著升高，并持续至实验结束。Ad-GDF 5治疗组椎间盘组织学改变较对照组明显改善。Ad-Luc注射组伤后2周开始糖胺聚糖（GAG）水平显著降低，术后4周DNA含量减少。相比之下，在注射Ad-GDF 5的椎间盘中，在整个8周治疗期间损伤后GAG和DNA水平没有降低。针刺小鼠椎间盘可建立椎间盘退变动物模型。腺病毒是基因递送的有效载体，具有靶蛋白的快速和延长的表达，并导致椎间盘退变标志物的改善。Ad-GDF 5基因治疗可以恢复受损椎间盘的功能，有可能成为一种有效的治疗方法。
The therapeutic strategies that have thus far been employed for the treatment of intervertebral disc degeneration (IDD) have focused on relieving the symptoms, while reversal of the degeneration remains an important challenge for the effective treatment of IDD. Growth and differentiation factor-5 (GDF5), of which deficiency leads to early disc degeneration changes, has the potential to increase proliferation of disc cells and expression of extracellular matrix proteins. To develop a lumbar disc degeneration model in mice and determine the effect of adenoviral GDF5 gene therapy. Compare the degeneration changes of discs punctured by different size needles to develop a mice lumbar disc degeneration model. Evaluate the effects of in vivo gene therapy for the mice disc degeneration model by an adenoviral vector carrying GDF5 gene. A lumbar disc degeneration model was developed by needle punctures to the discs in Balb/c mice. Afterwards, a gene therapy treatment to disc degeneration was evaluated. Two of the mice lumbar discs were randomly chosen to be punctured by a 30- gauge needle and then injected with adenovirus that had been engineered to express either the luciferase gene (Ad-Luc) or the GDF5 gene (Ad-GDF5). Animals were analyzed by bioluminescent imaging, radiographic and MRI scanning, then sacrificed at 1-, 2-, 4-, or 8- week post operation and subjected to histological and biochemical assays. By the detection of T2-weighted MRI scanning and histological study, the degeneration was found in all of the discs punctured by different size needles. But the development of the degeneration in the discs injured by 30-gauge needle was more reliable and moderate compared with other groups. The detection of luciferase activity by bioluminescent imaging revealed that adenovirus survived and the introduced genes were expressed over 6 weeks after injection. There were no T2-weighted MRI signals in either the Ad-Luc or Ad-GDF5 injected mice up to 4 weeks post operation. At 6 and 8 weeks, T2-weighted signals were detected in the Ad-GDF5 group, but none in the Ad-Luc control group. The percent disc height index (%DHI) was significantly decreased (~ 20%) by 1 week following injury in both groups, indicating the development of disc degeneration. At 2 weeks, the %DHI in the mice injected with Ad-GDF5 increased significantly compared with that of the mice injected with Ad-Luc group; the increase was sustained for the rest of experiment period. The disc histology treated with Ad-GDF5 was improved compared with that in control group. Glycosaminoglycan (GAG) levels were significantly decreased in the Ad-Luc injection group since 2 weeks after injury, and the DNA content had diminished by 4 weeks after the operation. In contrast, in the discs injected with Ad-GDF5, there was no decrease in the GAG and DNA levels following injury throughout the 8 weeks treatment period. Disc degeneration animal model can be developed by using needle puncture to the discs in mice. The adenovirus is an effective vehicle for gene delivery with rapid and prolonged expression of target protein, and resulting improvement in markers of disc degeneration. Ad-GDF5 gene therapy could restore the functions of injured discs and has the potential to be an effective treatment.
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