【作者】 沈昌焕；

【导师】 欧云生；

【作者基本信息】 重庆医科大学 ， 外科学， 2010， 硕士

【摘要】 椎间盘退变性疾病是骨科临床常见病、多发病,包括颈椎病、腰椎间盘突出症、腰椎管狭窄症等。免疫性炎症反应、髓核刺激压迫相邻神经和血管引起的相应症状、体征困扰着患者和医疗工作者。国内外临床MRI等影像学资料观察发现较低年龄群体中椎间盘就存在不同程度退变,但从临床资料分析来看,该类疾病的发病情况个体差异较大[1,2]。目前国内外对这类疾病的发病机理阐述并不完善,针对病因的预防性治疗的阐述甚少。虽然临床诊断方法、手术方法等技术在不断提高,但椎间盘疾患的治疗仍存在诸多问题如:保守治疗疗效不佳、手术治疗并发症多等等。这些促使研究者对椎间盘退变疾病的发病机理进行更加深入的研究。椎间盘是人体最大的无血供结构,其退变的特征是蛋白聚糖含量下降、水分减少、胶原纤维构成比改变等。新近的研究表明生物力学的作用是次要的,更强调生物化学机制在椎间盘退变中的作用[3,4],但启动和调节这一过程的生物学机制目前仍不十分清楚。细胞外基质(extra cell matrix,ECM)成分的变化可能是直接导致椎间盘退变的一个重要原因,基质金属蛋白酶(matrix metalloproteinase,MMPs)在椎间盘退变的启动和调节生物学机制中发挥着重要作用[5,6],同时MMPs相关基因的研究也使人们对椎间盘突出的病因有了更深入的认识[7,8]。MMPs是参与降解全身各种组织ECM的蛋白酶家族,目前椎间盘ECM中相关研究较多的MMPs包括MMP-1、2、3、7、9、13等。MMP-7又称基质溶解素,是MMPs家族中最小的独立分子,也是一个比较重要的成员,MMP-7在细胞外基质和基膜降解中起重要作用。MMP-13来源于软骨细胞,主要降解Ⅱ型胶原,还能降解聚集蛋白聚糖(aggrecan),从而在基质降解过程中发挥双重作用[9]。MMP-13可以受到体内多种细胞因子的调节,包括MMP-2、MMP-3等其它MMPs可以提高MMP-13的活性[10]。作为降解ECM的重要成分,MMPs在椎间盘退变中发挥的作用等相关研究显得非常重要。作为椎间盘重要营养途径结构的软骨终板的改变及MMPs的表达等研究也显得非常必要。通过研究不同程度退变椎间盘中MMP-7和MMP-13在软骨终板、髓核、纤维环不同结构中的表达与分布,了解椎间盘不同结构细胞、ECM在其退变过程中发挥的作用,探讨MMP-7和MMP-13在颈腰椎间盘退变中发挥的作用。方法:临床严格筛选颈椎病、腰椎间盘突出症、短期内脊柱外伤(椎间盘相对正常)患者,获得人不同程度退变椎间盘患者共32例,分为椎间盘突出组、椎间盘脱出组以及外伤性对照组。所有患者术前完善X线、MRI等辅助检查,使用视觉模拟评分法(visual analogue scale,VAS)评定患者疼痛程度。行手术治疗,术中切取患者病变椎间盘纤维环、髓核、软骨终板,分别用4%多聚甲醛固定约24小时后行脱水、包埋、切片等操作。通过HE染色、免疫组化、原位杂交方法检测椎间盘各部位MMP-7、MMP-13在细胞中的表达以及在ECM中含量的变化;通过免疫组化方法检测椎间盘各部位I型和II型胶原纤维含量变化,结合退变椎间盘标本的临床资料,分组统计学t检验分析实验结果。结果:(1)椎间盘突出患者的软骨终板和髓核中MMP-7和MMP-13均有较高表达,免疫组化半定量分析MMP-7在突出组软骨终板高于脱出组软骨终板(p<0.05);免疫组化单位面积阳性细胞表达均值MMP-7突出组软骨终板高于脱出组软骨终板(p<0.05)。(2)椎间盘脱出患者髓核细胞中MMP-7表达较高,免疫组化半定量分析MMP-7在脱出组髓核高于突出组髓核(p<0.05);免疫组化半定量分析MMP-13在脱出组软骨终板高于突出组软骨终板(p<0.05);免疫组化单位面积阳性细胞表达均值MMP-7和MMP-13脱出组髓核高于突出组髓核(p<0.05);免疫组化单位面积阳性细胞表达均值MMP-7脱出组软骨终板高于突出组软骨终板。(3)免疫组化分析突出组和脱出组椎间盘三个结构MMP-7和MMP-13均高于对照组。(4)原位杂交单位面积阳性细胞表达均值MMP-13突出组软骨终板高于脱出组软骨终板。结论:实验结果提示:(1)MMP-7和MMP-13在椎间盘退变过程中有高度表达,且基本与退变程度正相关。(2)MMP-7、MMP-13在不同退变程度、不同椎间盘结构中表达不一致。早期退变过程MMP-7在软骨终板较高表达,中晚期退变过程MMP-13在椎间盘软骨终板较高表达,提示软骨终板细胞可能在参与椎间盘不同时期退变过程中发挥不同作用。更多还原

【Abstract】 Degenerative disc disease is the orthopedic clinical common disease, including cervical spondylosis, lumbar disc herniation and the lumbar spinal stenosis. Immune inflammatory response, nerves and blood vessels stimulated and oppressed by the nucleoplasty cause the appropriate symptoms and signs troubled patients and surgeon. Domestic and foreign clinical MRI imaging data shows that in the lower age groups there are varying degrees of disc degeneration, but in terms of clinical data analysis, the incidence of such diseases, had large individual differences. In present the pathogenesis of these diseases are not well described, the cause of preventive treatment for little elaboration. Although clinical diagnostic methods, surgical methods in continuous improvement to the treatment of intervertebral disc disease, there are still many problems exist such as: poor efficacy of conservative treatment, surgery complications and so on. These prompted researchers to study disc degeneration disease pathogenesis for more in-depth research. The disc is the largest no blood supply of the human body structure, characterized by degeneration of proteoglycan content decreased, water reduction, collagen fibers content changing. Recent studies show the role of biomechanics is secondary; emphasize biochemics process in the role of disc degeneration. But the start and control of this biochemical process are not clear. Changes in extracellular matrix may be a direct result of disc degeneration. Matrix metalloproteinase plays an important role in intervertebral disc degeneration biochemical process of initiation and regulation, while MMPs-related genes also let people to understand the role of MMPs in the disc degeneration.MMPs are involved in ECM degradation protease family, current research in the disc ECM have more MMPs, including MMP-1, 2,3,7,9,13 and so on. MMP-7, also known as matrilysin, is the smallest MMPs family of independent elements and a more important member, MMP-7 in the extracellular matrix and basement membrane degradation plays an important role. MMP-13 from chondrocytes, not only degradeⅡcollagen degradation, but also degrade aggrecan, resulting in substrate degradation played a important role. MMP-13 can be increased the activity by a variety of cytokines of the body, including MMP-2, MMP-3 and other MMPs. As an essential component of ECM degradation protease, MMPs play a very important role in disc degeneration. As an important structure of disc sustain way, cartilage endplate change and the expression of cartilage endplate MMPs research is very necessary. By studying the different degrees of degenerative disc MMP-7 and MMP-13 in the cartilage endplate, nucleus pulposus, annulus fibrosus, learning the expression of different structure and distribution, to understand different disc structure of cells, ECM degeneration process in its role of MMP-7 and MMP-13 in the cervical and lumbar disc degeneration.Method:Strictly screening out clinical case of cervical spondylosis, lumbar disc herniation, spinal injury patients in the short term as varying degrees of human intervertebral disc degeneration in patients with a total of 32 patients and divided them into 3 group: disc prominent group, disc prolapse group and the trauma group. All patients had X ray, MRI or other clinical examinations, using the VAS assessed the pain of patients. Cut anulus fibrosus, nucleus pulposus, cartilage endplate, respectively, by surgical treatment. With 4% paraformaldehyde for about 24 hours underwent dehydration, embedding, slicing up and other operations. By HE staining, immunohistochemistry, in situ hybridization detection of disc MMP-7, MMP-13 expression and the content changes in the ECM. Detected disc and all parts of I-type II collagen content by immunohistochemistry, combined with degenerative disc cases of clinical data, studied statistical analysis of experimental results.Results: (1)MMP-7 and MMP-13 expression were high in cartilage endplate and nucleus pulposus of disc prominent group, semi-quantitative immunohistochemical analysis of MMP-7 in a prominent group of cartilage endplate above the prolapse group (p <0.05); positive cells per unit area by immunohistochemical expression of MMP-7 of prominent group cartilage endplate above the prolapse group (p <0.05). (2) MMP-7 expression were high in nucleus pulposus of disc prolapse group, semi-quantitative immunohistochemical analysis of MMP-7 in the prolapse group were higher than prominent group of nucleus pulposus (p <0.05); semi-quantitative immunohistochemical analysis of MMP-13 in the prolapse group cartilage endplate above prominent group (p <0.05); immunohistochemical positive cells per unit area MMP-7 and MMP-13 in prominent group nucleus pulposus were higher than prolapsed group (p <0.05); immunohistochemical expression of positive cells per unit area MMP-7 prolapse group cartilage endplate above the prominent group. (3) Immunohistochemical analysis of prominent groups and prolapse group of three structures MMP-7 and MMP-13 were higher than the trauma group. (4)Positive cells per unit area in situ hybridization, the expression of MMP-13 prominent group cartilage endplate above the prolapse group.Conclusion:The results suggest that: (1) MMP-7 and MMP-13 in the disc degeneration process are highly expressed, and associated with the basic degeneration. (2) The expression of MMP-7, MMP-13 in different degeneration and different disc structure are inconsistent. In early degeneration the MMP-7 expression in the cartilage endplate is high, in middle and late degeneration the MMP-13 expression of intervertebral disc cartilage is high, showing cartilage endplate cells may participate in the process of disc degeneration play different roles in different times.更多还原