【作者】 李想；

【导师】 王以朋；

【作者基本信息】 北京协和医学院 ， 外科学， 2011， 博士

【摘要】 [研究背景]我们实验室前期的研究结果显示,在青少年特发性脊柱侧凸(AIS)患者侧凸顶点终板软骨凸凹侧蛋白质组存在显著差异,这些存在表达差异的蛋白很可能与椎间盘的不对称发育有关。同时由于脊柱侧凸可导致椎间盘承受异常的应力,AIS患者椎间盘在成熟度和退变程度上与同龄正常人群存在显著差异,这些存在表达差异的蛋白也很可能参与了椎间盘退变的病理过程。软骨调节素(chondromodulin-Ⅰ, ChM-Ⅰ)是仅在凸侧表达的关键蛋白之一。其主要作用在与促进软骨生长,维持软骨及所在组织的无血管活性,并与骨关节炎、心脏瓣膜病以及骨、软骨肿瘤的发病有关。有关ChM-Ⅰ在椎间盘退变过程中的作用还未见深入报道。[目的]1.利用分子生物学及免疫组织化学方法研究ChM-Ⅰ在成人退变椎间盘细胞中的表达情况。2.观察碱性成纤维细胞生长因子(bFGF)对椎间盘细胞ChM-Ⅰ表达的影响。3.观察ChM-Ⅰ在不同退变程度椎间盘组织中的表达情况。4.对ChM-Ⅰ在椎间盘退变过程中可能发挥的作用作初步探讨。[方法]1.取3例因腰椎间盘退变性疾病而需行后路椎间融合患者的椎间盘组织分别进行髓核及纤维环细胞培养。取部分原代细胞利用RT-PCR和Western blot研究ChM-ⅠmRNA和蛋白在成人退变椎间盘细胞中的表达情况。2.利用不同浓度(0ng/ml, 1ng/ml和10ng/ml) bFGF刺激椎间盘细胞,利用real-time RT-PCR和Western-Blot研究不同浓度bFGF对髓核细胞和纤维环细胞ChM-ⅠmRNA和蛋白表达的影响。3.收集因腰椎间盘退变性疾病而行手术切除的椎间盘组织标本28例,根据MRI表现退变程度为Ⅲ-Ⅴ级,作为退变组。收集因脊柱肿瘤行手术治疗时切除的椎间盘标本6例,退变程度Ⅰ级,作为对照组。利用免疫组织化学方法研究ChM-Ⅰ在不同退变程度椎间盘组织中的表达情况。[结果]1. RT-PCR和Western blot结果显示ChM-I在椎间盘髓核细胞及纤维环细胞中均有表达,两者间在表达程度上无显著差异。2. bFGF可明显抑制ChM-I mRNA和蛋白在髓核细胞及纤维环细胞中的表达,且呈剂量依赖性(P<0.05)。3. ChM-I在对照组(无退变椎间盘组织)表达量很低,而在椎间盘发生退变后其表达量明显升高,ChM-I在不同退变程度椎间盘组织中的表达存在显著差异(P<0.05)。[结论]1.首次利用RT-PCR和Western blot方法证实髓核及纤维环细胞可表达ChM-I。2.髓核细胞和纤维环细胞中ChM-I的表达受bFGF的影响。3.椎间盘发生退变后ChM-I表达明显升高,提示其可能作为一种防御机制参与了椎间盘退变的病理过程。更多还原

【Abstract】 Background Previous study of our laboratory showed that there were different protein expression levels between the convex and concave sides of the intervertebral discs endplate cartilage in AIS patients. The key proteins which express on the convex or concave side exclusively may involved in the progress of the AIS. Recent study showed that morphologic disc degeneration was accelerated about 20 years in scoliosis versus physiological ageing. The result indicated that the key proteins may also involved in the progress of intervertebral disc degeneration. Chondromodulin-I (ChM-I) is one of the key proteins which express on the convex side exclusively. ChM-I can stimulate the proliferation of chondrocytes and maintain the avascularity and anti-angiogenic properties of normal cartilage. It is also associated with the progress of osteoarthritis, valvular heart diseases and tumor. Yet the role of ChM-I in intervertebral disc degeneration is still unkown.Objectives1. To investigate the expression of ChM-I in the intervertebral disc cells.2. To investigate the effect of bFGF on the expression of ChM-I in the intervertebral disc cells.3. To examine the expression of ChM-I and to correlate its expression with the degree of disc degeneration.4. To discus the role of ChM-I in the disc degeneration.Methods1. Three IVDs obtained from patients in the treatment of disc degenerative disease were used for cell culture. ChM-I expression in IVD cells was examined by using RT-PCR and Western blot.2. The effect of bFGF on the expression of ChM-I was assessed by real-time PCR and Western blot respectively.3. Twenty-eight human IVD tissues were obtained from patients in the surgical treatment of disc degenerative disease at different stage of degeneration according to MRI.6 IVD tissues removed from patients with metastatic spinal tumor were used as normal control. The expression of ChM-I determined by immunohistochemical analyses was correlated with MRI degeneration grade.Results1. RT-PCR and Western blot examination showed that ChM-I expressed in both adult degenerative anulus fibrosus cells and nucleus pulposus cells.2. The mRNA and protein expression of ChM-I were both down-regulated by administration of bFGF with dose-dependent way(P<0.05)3. Immunohistochemical analyses showed the number of ChM-I-expression cells in human IVD tissues was significantly elevated in advanced stages of IVD(grade III, IV, V) degeneration compared with those in normal control(P<0.05)Conclusions1. ChM-I mRNA and protein are expressed in IVD cells.2. The expression of ChM-I is down-regulated by administration of bFGF.3. The expression of ChM-I in discs with advanced stage of degeneration is elevated compared with those with nondegeneration which indicate that the expression of ChM-I is upregulated through a defense mechanism against the degenerative process of intervertebral disc.更多还原