【作者】 刘斌；

【导师】 金大地；

【作者基本信息】 第一军医大学 ， 外科学， 2004， 博士

【摘要】 研究背景： 椎间盘退行性病变常可导致原发性下腰痛、颈腰神经根病等多种临床综合征，临床上椎间盘退行性疾病十分常见，严重影响了人们的健康。虽然目前对椎间盘退变的病因尚不完全了解，但系列研究证实，软骨终板是椎间盘的重要营养途径，椎间盘退变与软骨终板退变密切相关，软骨终板退变可能是导致椎间盘退变的一种主要因素。而目前对软骨终板退变研究多局限于形态学等方面，对其发病机理的研究甚少。目前研究发现，炎性细胞因子、NO和细胞凋亡等因素在椎间盘退变及关节软骨退变过程中发挥重要促进作用，而一些生长因子如TGF—β₁、IGF-Ⅰ等可以拮抗IL-1β、IL-6、TNF-α等炎性细胞因子、NO和细胞凋亡对关节软骨的破坏作用。由于软骨终板结构组成和生化特性与关节软骨相似，因此推断，这几种因素在软骨终板退变中同样发挥着重要作用。通过研究以上各种因素在软骨终板退变中的作用，探讨它们之间作用的相互关系，明确软骨终板退变的启动因素及主要因素，并通过应用生长因子，试图延缓和阻止软骨终板退变的过程，为椎间盘退变的治疗提供新的思路和途径。 目的： （1）通过造成兔腰椎椎间失稳，诱导软骨终板退变发生，探讨软骨终板组织结构及生化组成方面的变化。 （2）通过体外培养软骨终板细胞，探讨不同因素对软骨终板细胞的作用，以明确定各种因素相互作用的关系。 （3）对软骨终板及细胞施加生长因子，探讨生长因子延缓和阻止软骨终板退变的作用。 方法： （l）选用新西兰白兔36只，随机分为手术组18只，对照组18只。手术组沿Ll一L:棘突做后正中切口，剥离椎旁肌，切除Ll一L:的棘上、棘间韧带及两侧关节突关节后外1/2;对照组仅切开皮肤后缝合。于术后12w、24w和36w进行腰椎X线检查、扫描电镜观察、组织学观察、No含量和Nos活性检测、IL一lp、IL一6、TNF一a含量检测、蛋白多糖含量及胶原释放量的测定。 （2）长枫杂交猪8只，进行软骨终板细胞原代培养，通过MTT比色法绘制细胞生长曲线，通过HE染色、甲苯胺蓝染色、番红O染色及11型胶原免疫细胞化学染色对软骨终板细胞进行鉴定;并分别应用硝普钠刺激软骨终板细胞，放射免疫法检测IL一lp、IL一6、TNF一a含量;PI、AO荧光染色法及DNA片段凝胶电泳法检测细胞凋亡;硝酸还原法检测NO含量;355掺入法检测蛋白多糖合成;3H一脯氨酸掺入法检测胶原合成。 （3）按不同时间段，将上述实验动物处死取材，进行组织培养，在施加上述因素同时施加TGF一pl、IGF一I、TGF一pl+IGF一I处理;并对软骨终板细胞亦在施加上述因素同时进行TGF一pl、IGF一I处理。进行NO含量和Nos活性检测，IL一lp、IL一6、TNF一。含量测定，细胞凋亡检测，蛋白多糖含量及胶原释放量的测定。结果: （1）随时间延长，实验组软骨终板与对照组相比钙化严重，胶原纤维排列无规则、纤维变细、断裂，蛋白多糖丢失明显;软骨终板变薄，潮标前移明显，血管芽周围钙化，血管芽数量减少:NO含量和NOS活性升高明显;IL一lp、IL一6、TNF一a含量亦随时间延长而增加，而蛋白多糖含量及胶原释放量则显著降低。 （2）软骨终板细胞经过不同染色鉴定后，可确定其具有软骨细胞特性;施加NO后，细胞发生凋亡，炎性细胞因子含量升高，蛋白多糖及胶原合成量下降;施加IL一lp、IL一6、TNF一a后，发现IL一lp和rrNF一a可明显增加NO含量，并促进细胞凋亡的发生，而IL一6则无此特性;但三种细胞因子均可降低蛋白多糖和胶原合成量;施加HZO:诱导出凋亡后，NO含量、炎性细胞因子含量、蛋白多糖及胶原合成量均明显下降。 （3）施加TGF一日1、IGF一I后，软骨终板及软骨终板细胞的IL一lp、IL一6、TNF一a含量明显降低;No含量和Nos活性显著降低;软骨终板细胞凋亡率降低，且IGF一I作用较TGF一pl显著;蛋白多糖及胶原生成量显著升高，且TGF一pl和IGF一I的协同作用比单独应用TGF一pl、IGF-I更强。结论: （l）腰椎动、静力平衡破坏可诱导软骨终板发生退变。腰椎失稳可造成软骨终板异常钙化，胶原形态发生改变。软骨终板退变可导致蛋白多糖、胶原含量降低，No含量和Nos活性增加，细胞凋亡增加，IL一lp、IL一6、TNF一a含量升高。 （2）IL一lp和TNF一a导致NO增加，NO可诱导细胞发生凋亡，细胞凋亡后，蛋白多糖和胶原生成量均降低;IL一6亦可导致蛋白多糖和胶原生成量均降低;NO增加会进一步导致IL一1旦、IL一6、TNF一。含量增加;但细胞凋亡会抑制NO和IL一lp、IL一6、TNF一a的生成。 （3） TGF一pl和IGF一I可减少炎性细胞因子的生成，同时也可直接减少NO的生成，通过NO途径抑制细胞凋亡，促使蛋白多糖及胶原含量升高，可延缓和阻止软骨终板退变。更多还原

【Abstract】 BackgroundDisc degeneration disease usually results in primary low back pain and cervical or lumbar radiculopathy syndrome. In clinical, it is a very commonly disease, which has dramatic effect on people’s health. The etiology of disc degeneration is still unclear, but series of research have showed that CEP degeneration may be the major cause of disc degeneration. Presently, the study of CEP degeneration is mostly limited to its morphologic change, however, the investigation of its etiology is too little. At present, it has been found that inflammatary cytokine?NO and apoptosis play an important role on the degeneration of disc and articular cartilage. While, several growth factor such as TGF-β?IGF- I can resist the role of inflammatary cytokine(IL-1β, IL-6, TNF-α), NO and apoptosis, which are easy to destroy the cartilage. The structure and biochemical characteristics of CEP is similar to article cartilage, therefore, it can be deduced that those factors may play the same role on CEP. With the study of those factors’ role on disc degeneration, the relationship to them is expected to know and the main factor is expected to be clear. By using the growth factors, the progress of CEP degeneration tried to be retarded or prevented in order to provide newly methods to the treatment of disc degeneration.Objective(1) To explore the structure and biochemical changes of CEP in the induced degeneration by injuring the intervertebral stability of the lumbar spine. (2) To evaluate the role of different factors on the chondrocytes of CEP so as to define the interaction of those factors by the culture of chondrocytes in vitro. (3) To investigate the effect of retardment or prevention of growthfactors on CEP degeneration by applying growth factors to CEP and chondrocytes.Methods(1) 36 New Zealand rabbits were divided into experimental group and control group randomly. The experimental group model of lumbar CEP degeneration in rabbits was established by resection of all lumbar supraspinous and interspinous ligaments, excision of parts of zygapophysial joints and detachment of the posterior paravertebral muscles from lumbar vertebraes. The control group model was incised only the skin. All of the rabbits examined 12w?24w and 36w after operation by X-ray, scanning electron microscope, histopathologic study, the detection of content of NO and the activity of NOS, the content of IL-1, IL-6 and TNF-a, the detection of content of proteoglycan and the release of collagen.(2) The chondrocytes were isolated and harvested from the CEP of 8 porcines, The growth curve was drawn by MTT chromatometry. The biological phenomena was observed by light microscope and HE staining. Their biological characteristics were identified by toluidine blue and safranine O staining for proteoglycan and immunocytochemistry for type II collagen. After using sodium nitroprusside (2mmol/L) , IL-lp(10ng/ml), IL-6(50ng/ml), TNF-a(50ng/ml) and H2O2(200mol/l) respectively to stimulate chondrocytes, the content of IL-1?IL-6?TNF-a was measured by radioimmunoassay. The apoptosis was observed by PI or AO fluorescent staining and DNA fragment gel electrophoresis. The content of NO and the activity of NOS were measured by Griess reaction method. The proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate Na235SO4. The incorporation of 3H-proline was used to detect synthesis of collagen.(3) According to different time, the CEPs were achieved from the above animals after death. And then, the tissues were cultured with the growth factor such as TGF-1?IGF- I and TGF-1?with IGF- I . TGF- 1 or IGF-I was also applied to the chondrocytes of CEP. Then, with using the previous methods, the activity of NOS, the content of NO, the content of IL-1, IL-6, TNF-a and proteoglycan and the release of collagen were detected.Results(1) With the elapse of time, the CEP of experimental group had more serious calcification than that of the control group. The collagen fibers became thin and breakdo更多还原