Quantitative analysis of gene expression in a rabbit model of intervertebral disc degeneration by real-time polymerase chain reaction.
Quantitative analysis of gene expression in a rabbit model of intervertebral disc degeneration by real-time polymerase chain reaction.
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DOI：
10.1016/j.spinee.2004.05.251
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发表时间：
2005-01-01
期刊：
影响因子：
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通讯作者：
Kang, James D
中科院分区：
文献类型：
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作者：
Sobajima, Satoshi;Shimer, Adam L;Kang, James D
背景：椎间盘退变（IDD）过程中基因表达的系列分析可以阐明对病理生理学的有价值的见解，并为开发新的细胞和基因治疗提供潜在靶点的鉴定基础。然而，以往很少有研究通过合适的动物模型来描述IDD过程中基因表达的变化。目的：利用最近开发的兔环状刺伤模型和实时逆转录聚合酶链反应（RT-PCR）技术，定量分析正常和刺伤椎间盘中选定细胞外基质（ECM）产物、分解代谢、合成代谢和抗分解代谢因子编码的关键兔特异性mRNA序列的表达变化。研究设计：采用实时RT-PCR技术对缓慢进展且可重复的IDD动物模型进行基因表达分析。方法：12只家兔用16号针穿刺L2-L3、L3-L4和L4-L5椎间盘，3只家兔作为假对照。在磁共振成像（MRI）扫描确认退行性改变后的第3、6、12和24周，从被刺椎间盘上采集髓核组织。采用兔特异性引物对1)细胞外基质（ECM）组成基因：胶原Ia型、IIa型和聚集蛋白进行实时荧光定量pcr；2)分解代谢基因：基质金属蛋白酶-3 （MMP-3）、诱导型一氧化氮合酶（iNOS）、白细胞介素-1 β (il -1 β)；3)合成代谢生长基因：骨形态发生蛋白-2、-7 （BMP-2、-7）、转化生长因子- β 1 （tgf - β 1）、胰岛素样生长因子-1 (IGF-1)；4)抗分解代谢基因：组织金属蛋白酶-1 （TIMP-1）抑制剂。这些数据被归一化为甘油醛磷酸脱氢酶（GAPDH）的mRNA水平，这是一个组成性表达基因。结果：在24周的研究期间，MRI图像证实髓核高T2信号区和被刺椎间盘信号强度进行性下降，与IDD一致。ECM组分、聚集蛋白和IIa型胶原mRNA水平在第3周明显下降，且从未恢复，而Ia型胶原mRNA在整个退变过程中逐渐升高。BMP-2、BMP-7和IGF-1 mRNA在第3周至第6周相对降低，但在第12周和第24周升高至接近术前水平。TIMP-1的表达在第3周急剧下降到术前水平的十分之一左右，并在整个退行性过程中保持低水平。其余的结果，包括tgf - β 1和分解代谢基因（MMP-3, il -1 β, iNOS）显示出双峰特征。基因表达在第3周增加，在第6周和第12周下降到较低水平，然后在第24周出现第二次高峰。结论：ECM成分以及合成代谢、分解代谢和抗分解代谢因子的基因表达谱显示出许多与人类椎间盘退变相似的特征，表明椎间盘（IVD）无法对损伤产生早期合成代谢反应，从而为椎间盘缺乏修复能力提供了可能的解释。分解代谢基因在退行性变的早期和晚期都被强烈上调，这有力地支持了合成代谢或分解代谢失衡在IDD中起主要作用的假设。根据所得到的模式，通过基因转移技术增加BMP-2、BMP-7、IGF-1或TIMP-1的早期产生可能会改变刺痛模型中所见的退化的进展过程。下一步将是转移这些治疗基因来调节生物过程，理想地改变椎间盘退变的进展过程。
BACKGROUND CONTEXT: Serial analysis of gene expression during the course of intervertebral disc degeneration (IDD) could elucidate valuable insight into pathophysiology and provide a basis for identification of potential targets for the development of novel cellular- and gene-based therapies. However, very few previous studies described the changes in gene expression through the process of IDD using a suitable animal model.PURPOSE: To use a recently developed rabbit annular stab model and the technique of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify the change in expression of key rabbit-specific mRNA sequences encoding for selected extracellular matrix (ECM) products, catabolic, anabolic, and anti-catabolic factors in normal and stabbed discs.STUDY DESIGN: Gene expression analyses were performed to characterize a slowly progressive and reproducible animal model of IDD using real-time RT-PCR.METHODS: Twelve rabbits underwent an annular stab with a 16-gauge needle to the L2-L3, L3-L4, and L4-L5 discs, and three rabbits served as sham controls. Nucleus pulposus tissues were harvested from the stabbed discs at 3, 6, 12 and 24 weeks after confirmation of degenerative changes by magnetic resonance imaging (MRI) scan. Real-time RT-PCR was performed with the use of rabbit-specific primers for 1) extracellular matrix (ECM) component genes: collagen type Ia and IIa, and aggrecan; 2) catabolic genes: matrix metalloprotease-3 (MMP-3), inducible nitric oxide synthase (iNOS), and interleukin-1beta (IL-1beta); 3) anabolic growth genes: bone morphogenic protein-2, and -7 (BMP-2, -7), transforming growth factor-beta1 (TGF-beta1), and insulin-like growth factor-1 (IGF-1); and 4) anti-catabolic gene: tissue inhibitor of metalloprotease-1 (TIMP-1). These data were normalized to mRNA levels of glyceraldehyde phosphate dehydrogenase (GAPDH), a constitutively expressed gene.RESULTS: The MRI images confirmed progressive decline in the nucleus pulposus area of high T2 signal and in the signal intensity of the stabbed discs over the 24-week study period consistent with IDD. The ECM components, aggrecan and collagen type IIa mRNA levels had decreased markedly by week 3 and never recovered, whereas type Ia collagen mRNA gradually increased throughout course of degeneration. BMP-2, BMP-7 and IGF-1 mRNA were relatively decreased from weeks 3 to 6 but then increased at weeks 12 and 24 to end at a level near the preoperative level. The TIMP-1 expression fell dramatically to approximately one tenth of the preoperative level by week 3 and remained low throughout the degenerative process. The remaining results, including those from TGF-beta1 and the catabolic genes (MMP-3, IL-1beta, iNOS) demonstrated a double peak characteristic. The gene expression increased by week 3, decreased to a low level at weeks 6 and 12 and then had a second, late peak at 24 weeks.CONCLUSIONS: The gene expression profiles of ECM components and anabolic, catabolic, and anti-catabolic factors demonstrate many characteristics similar to the findings in human disc degeneration and suggest an inability of the intervertebral disc (IVD) to mount an early anabolic response to injury, thereby offering a possible explanation for the disc's lack of reparative capabilities. Catabolic genes are strongly up-regulated both early and late in degeneration, lending strong support to the hypothesis that an anabolic or catabolic imbalance plays a primary role in IDD. According to the resultant patterns, augmenting early production of BMP-2, BMP-7, IGF-1 or TIMP-1 by gene transfer techniques might possibly alter the progressive course of degeneration as seen in the stab model. The next step will be to transfer these therapeutic genes to regulate the biologic processes and ideally alter the progressive course of disc degeneration.