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Molecular analysis for the mechanisms of endoplate sclerosis using laser microdissection technique

利用激光显微切割技术对内板硬化机制进行分子分析

基本信息

批准号：
18591623
负责人：
KOGA Daisuke
金额：
$ 2.51万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

The pathogenesis of intervertebral disc degeneration is poorly understood, but is known to be associated with a variety of cellular and biochemical changes. Vertebral endplate changes and bone marrow changes visible in magnetic resonance imaging (MRI), are closely associated with dipc degeneration. To investigate molecular mechanisms underlying the process of endplate degeneration, we tried to apply laser capture microdissetion combined with microarray analysis to vertebral endplate.Although we have succeeded in extract total RNA from vertebral endplate, its quantity has not been enough for microarray analysis. Thus, we could not help abandon carry out the microarray analysis. However, we could acquire many skills for the analysis of hard tissues including bone and cartilage. Besides, we have developed novel disc degeneration model for rat tail.We have already developed continuous weight bearing disc degeneration model for mouse tail. In this model, degradation of IVD was observed with … More in two weeks after operation. Using this model, we tested if weight loading, a most common cause of intervertebral disc (IVD) degeneration, induces Runx2 expression, which may cause chondrocyte hypertrophy. Indeed, weight loading of the IVD by compression for a day significantly increased Runx2 expression. Thus, we next examined the expression of Runx2 with other disc degradation markers in canine IVD, The expression of RUNX2, type-X collagen and MMP-13 mRNA in young intact and degenerated IVD were examined by semi-quantitative RT-PCR analysis. The localization of Runx2 and type-X collagen protein in control and degenerated IVD were examined by imunohistochemistly. The expression of Runx2 transcript and protein, in combination with type-X collagen and MMP-13, was enhanced in degenerated IVDs of the dog.Next, we have investigated the relationship between expression patterns of Runx2 and the degree of intervertebral disc degeneration in canine NP cells through comparing the expression patterns to MMP-13 expression, a disc degeneration marker, and to MRI findings. Canine disc specimens were obtained from chondrodystrophoid dogs, including Dachshund and Beagle. RACE method indicated the canine Runx2 cDNA showed over 97% conservation with human RUNX2 and 95% with mouse. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that mRNA levels of Runx2 and MMP-13 were similarly increased in degenerated NP and herniated specimens. Immunohistochemical analysis indicated that T2/CSF ratio in MRI images was significantly correlated with Runx2 protein expression rate in various age beagle disc samples. Thus, our findings suggest Runx2 is a novel marker for disc degeneration.As mentioned above, intervertebral disc degeneration induced by mechanical compression is an important issue in spinal disorder research. In this study, the biomechanical aspect of the rat tail model was also investigated. An external loading device equipped with super-elastic TiNi springs was developed to apply a precise load to the rat tail. By using this device, rat tail discs were subjected to compressive stress of 0.5 or 1.0 MPa for 2 weeks. Discs in the sham group received an attachment of the device but no loading. After the experimental period, first the intact tail with peripheral tissues (PT) such as tendon and skin and then the retrieved disc without PT were subjected to a uniaxial tension-compression test ; biomechanical characteristics such as range of motion (ROM), neutral zone (NZ), and hysteresis loss (HL) were evaluated. Furthermore, the loadbearing contribution of PT in the intact tail was estimated by comparing the load-displacement curves obtained by the mechanical tests performed with and without PT.The experimental findings revealed that the continuous compressive stress induced reduction in disc thickness. The intact tail demonstrated decreases in ROM and NZ as well as increases in HL.On the other hand, the retrieved disc demonstrated increases in ROM, NZ, and HL.Further, a significant increase in the load-bearing contribution of PT was indicated. These findings suggest that the load-bearing capacity of the disc was seriously deteriorated by the application of compressive stress of 0.5 or 1.0MPa for 2 weeks.Lastly, we have developed novel LCM technique applied for hard tissues, such as articular cartilage. Using our techniques, we have been investigating the mechanisms of cartilage degeneration. Less

椎间盘退变的发病机制知之甚少，但已知与各种细胞和生化变化有关。在磁共振成像（MRI）中可见的椎体终板变化和骨髓变化与dipc变性密切相关。为了探讨终板退变的分子机制，我们尝试将激光捕获显微切割技术与基因芯片技术相结合，从椎体终板中提取总RNA，但其数量尚不足以用于基因芯片分析。因此，我们不得不放弃进行微阵列分析。然而，我们可以获得许多技能，用于分析硬组织，包括骨和软骨。此外，我们还建立了一种新的大鼠尾椎间盘退变模型，我们已经建立了小鼠尾椎间盘持续负重退变模型。在该模型中，观察到IVD降解， ...更多信息术后2周。使用该模型，我们测试了体重负荷（椎间盘（IVD）退变的最常见原因）是否诱导Runx 2表达，这可能导致软骨细胞肥大。事实上，通过压缩一天的IVD的重量负荷显著增加Runx 2表达。因此，我们接下来检测了Runx 2与其他椎间盘降解标志物在犬IVD中的表达。通过半定量RT-PCR分析，检测了RUNX 2、X型胶原和MMP-13 mRNA在年轻完整和退化IVD中的表达。免疫组化法检测Runx 2和X型胶原蛋白在正常和退变IVD中的定位。Runx 2的转录和蛋白的表达，结合X型胶原和MMP-13，增强在退化的IVDs的狗。接下来，我们研究了Runx 2的表达模式和椎间盘退变的程度之间的关系，在犬NP细胞通过比较表达模式MMP-13的表达，椎间盘退变的标志物，和MRI结果。犬椎间盘标本取自软骨营养不良犬，包括腊肠犬和比格犬。RACE方法显示犬Runx 2基因与人Runx 2基因的保守性在97%以上，与小鼠Runx 2基因的保守性在95%以上。逆转录聚合酶链反应（RT-PCR）分析显示，Runx 2和MMP-13的mRNA水平在退化的NP和疝出标本中同样增加。免疫组化分析表明，T2/CSF在MRI图像的比例与Runx 2蛋白表达率在不同年龄的比格犬椎间盘标本显着相关。因此，我们的研究结果表明Runx 2是一个新的椎间盘退变的标志物。如上所述，机械压迫引起的椎间盘退变是脊柱疾病研究的一个重要问题。在这项研究中，生物力学方面的大鼠尾部模型也进行了研究。研制了一种采用超弹性TiNi弹簧的外加载装置，用于对大鼠尾部施加精确的载荷。通过使用该装置，使大鼠尾盘经受0.5或1.0MPa的压应力2周。假手术组的椎间盘接受了器械的附件，但没有加载。实验期结束后，首先对具有周围组织（PT）（如肌腱和皮肤）的完整尾部，然后对无PT的回收椎间盘进行单轴拉伸-压缩试验;评价生物力学特性，如活动度（ROM）、中性区（NZ）和滞后损失（HL）。此外，在完整的尾部PT的承载贡献估计通过比较载荷-位移曲线进行了机械测试与PT。实验结果表明，连续的压缩应力诱导减少盘的厚度。完整的尾部表现出ROM和NZ的减少以及HL的增加。另一方面，回收的椎间盘表现出ROM，NZ和HL的增加。此外，PT的承重贡献显着增加。这些结果表明，施加0.5或1.0MPa的压应力2周后，椎间盘的承载能力严重恶化。最后，我们开发了适用于硬组织（如关节软骨）的新型LCM技术。使用我们的技术，我们一直在研究软骨退化的机制。少