【作者】 刘超；

【导师】 范顺武；

【作者基本信息】 浙江大学 ， 骨科学， 2011， 博士

【摘要】 研究背景：椎间盘退变可能是多因素协同作用的结果,其中营养因素是不可忽视的原因之一。作为全身最大的无血管组织,任何对椎间盘周围毛细血管网产生的干扰都是对椎间盘营养供给的潜在危险因素和椎间盘退变的可能易感因素。糖尿病不但引起体内代谢紊乱,而且极易诱发周围微血管病变。因此有学者认为糖尿病可能也是椎间盘退变的影响因素之一。糖尿病同时伴发下腰痛患者往往在日常生活和治疗过程中承受更大的痛苦和受到更多的限制。尽管有少量文献报道,但是关于糖尿病与椎间盘和终板退变的相关性及其具体机制并未深入研究。此外,糖尿病时椎间盘细胞外基质中的主要成分以及影响代谢的相关因子的变化情况仍不清楚。研究目的：1.通过临床影像学观察,研究糖尿病对椎间盘退变的影响,分析椎间盘和终板Modic退变在下腰痛伴或不伴糖尿病患者中分布的异同;2.通过观察经典的糖尿病大鼠模型,利用影像学和组织学方法研究糖尿病状态时椎间盘退变的情况,探讨糖尿病大鼠模型是否能作为一种新的研究椎间盘退变疾患的动物模型：3.通过研究糖尿病大鼠椎间盘细胞外基质的变化,深入探讨糖尿病对水分、胶原和蛋白多糖,以及与之相关的代谢相关因子的影响,同时进一步研究这些代谢因子的变化在糖尿病椎间盘退变中的意义。研究方法：1.回顾性分析2007年～2010年以下腰痛为主述来我科行手术治疗的患者127例,以及同期至我院行其它择期手术患者145例,比较糖尿病在两组患者间的构成比；通过影像学MR1分析和比较椎间盘退变和终板Modic退变在糖尿病和非糖尿病患者中的比例和分布情况；对下腰痛患者进行跟踪随访,对患者术前术后VAS评分和ODI评分进行评价；2.取20只SD大鼠,利用STZ诱导辅以高糖高脂饮食制作糖尿病大鼠模型,另取20只SD大鼠做对照；对模型进行不同时间点(造模前、4周、8周和12周)的影像学研究,分析X线上椎间盘高度指数和T2加权像MRI椎间盘信号强度的变化；同期取部分样本做HE常规染色和Safranin O染色,在排除其它可能致椎间盘退变偏倚因素的基础上,分析糖尿病在动物模型上对椎间盘退变的影响;3.对糖尿病状态时椎间盘细胞外基质主要成分的分布与含量进行生化学研究,包括测定髓核水分含量,DMMB)去测定GAG含量,和免疫印迹测定Ⅰ型、Ⅱ型胶原以及aggrecan蛋白的含量变化；利用实时定量PCR对椎间盘代谢相关因子MMP-3、BMP-2、TIMP-1的mRNA水平进行定量分析。研究结果：1.在下腰痛患者中糖尿病的构成比明显高于其它择期手术患者,差异有统计学意义(X2=16.17,P<0.01)；糖尿病患者并发其它严重合并症,特别是高血压(P<0.01)和骨质疏松(P<0.05)的比例也明显高于非糖尿病患者;椎间盘退变Ⅲ级在非糖尿病患者中比例明显较糖尿病患者多(P<0.05),但是椎间盘退变Ⅴ级在糖尿病患者中明显增多(P<0.05)Modic退变2型在糖尿病患者中分布明显多于非糖尿病患者(P<0.05)；医师测评糖尿病与非糖尿病患者手术优良率并无差异：两者在术前术后VAS评分和ODI改善率上并无明显差别(P>0.05)：2.STZ诱导辅以高糖高脂饮食可以成功复制糖尿病大鼠模型,在观察期内实验组大鼠平均血糖值仍保持在16.7mmol/L以上,具有良好的模型稳定性；影像学X线观察发现实验组大鼠造模后4周椎间隙开始出现狭窄,椎体逐渐失稳,第12周时椎间隙狭窄明显,并出现骨赘；与同期对照组相比,第8周和第12周实验组椎间隙明显狭窄,结果有显著性差异(P<0.01)。T2加权像MRI上随时间的延长实验组大鼠椎间盘信号降低的趋势越明显,与同期对照组相比,椎间盘信号指数有显著性差异(P<0.05)；同期HE染色和Safranin O染色也证明椎间盘有明显的退变,这些改变与影像学有较好的相关性；3.糖尿病椎间盘的细胞外基质中,水分含量在实验组椎间盘中逐渐减少,第8周和第12周时分别为(68.2±6.2)%和(60.1±9.7)%,显著少于同期对照组(P<0.05,P<0.01)；实验组GAG含量总体呈下降趋势,在第8周时显著少于对照组(P<0.05)；免疫印迹发现实验组大鼠椎间盘中aggrecan蛋白的表达量随着时间的推移而逐渐下降,到第8周时表达最低,而第12周时其表达又略有增加；Ⅰ型胶原蛋白表达自第8周开始,在糖尿病模型大鼠椎间盘组织中有较高的表达,并且一直持续到第12周；Ⅱ型胶原蛋白的表达则逐渐减少,至第12周时最低；实时定量PCR结果发现,实验组MMP-3在8周时(?)nRNA水平表达明显增高,与同期对照组相比有显著差异(P<0.05),并且之后继续升高,至第12周时为(8.5±1.3)倍(P<0.01)；BMP-2在第4周时表达升高至(5.4±2.1)倍,与对照组相比结果有显著差异(P<0.05),但此后又呈下降趋势,至第12周时已降至造模前的水平TIMP-1仅在第12周时有显著高表达(P<0.05)。研究结论：1.糖尿病是椎间盘退变的影响因素之一；糖尿病患者合并其它并发症的比例明显增高；椎间盘退变的终末期与Modic退变2型比例在糖尿病患者中较多；但是糖尿病与非糖尿病患者手术疗效并无差别;2.以STZ辅以高糖高脂诱导的糖尿病大鼠模型具有良好的稳定性,可以作为研究糖尿病与椎间盘退变关系的动物模型；糖尿病状态时大鼠椎间盘退变的进展加速：3.糖尿病状态时椎间盘细胞外基质中主要成分水分和aggrecan蛋白含量减少,Ⅰ型/Ⅱ胶原比例增加,椎间盘内代谢紊乱,代谢相关因子的异常变化很可能是造成细胞外基质合成和降解紊乱并导致椎间盘退变的影响因素。更多还原

【Abstract】 Background:Disc degeneration is one of the most causes of low back pain, which is the result of multiple factors, including nutritional factor. As the largest avascularity tissue in the human body, anything induced destroys of capillary network around the intervertebral disc will be the risk factor of nutritional supply and the predisposing factor of disc degeneration. Diabetes mellitus is a metabolic disease inducing not only the metabolic disturbance, but also microangiopathy. For that reason, some authors suggested that diabetes mellitus might be an influencing factor of disc degeneration. The coexistence of diabetic and lumbar spine disease may cause even greater limitation among the diabetic population when compared with nondiabetic one, prompting more aggressive treatment. Although there was few literature. the interactions between diabetes mellitus and the degenerations of intervertebral disc and endplates was still uncertain. Furthermore, the changes of extracellular matrix and metabolic factors of intervertebral discs in diabetes mellitus are still unknown.Objective:1. To study the influence of diabetes mellitus on disc degeneration Via clinical observation. and to discuss the relationship between diabetes mellitus and the degenerations of intervertebral disc and endplates: 2. To study the disc degeneration in classical diabetic rat model via radiology and histology, and to discuss whether the classical diabetic rat model could be a new animal model to study the disc degeneration;3. To study the changes of extracellular matrix in the intervertebral discs of rats, including the contents of water, collagen, and proteoglycan, and to study the interaction with the metabolic factors, for example, MMP-3, BMP-2 and TIMP-1. Methods:1. Baseline characteristics of 127 patients with low back pain were compared with those of selective operations for other chief complaints in a total of 145 patients in the constituent ratio:the rate and distribution of disc and endplate degenerations in the coexistence diabetic and low back pain patients compared with those without diabetes mellitus; the operative outcomes including VAS and ODI were evaluated between patients with or without diabetes mellitus;2. The study group including 20 SD rat was induced by STZ and both high glucose and fat diet for diabetic animal model:the disc height index on X ray and the signal intensity on T2-weighted MRI were examined at different time points (baseline.4-.8-. and 12-week):the histological examination including HE and Safranin O stains were evaluated at the same time, in order to demonstrate the influences of diabetes mellitus on disc degeneration in the animal model;3. The extracellular matrix in the intervertebral disc was evaluated, including the contents of water and GAG measured by DMMB. the changes of types I and II collagen, and the aggrecan measured by western blot: the mRNA levels of MMP-3. BMP-2 and TIMP-1 was evaluated by qPCR. Results:1. The constituent ratio in low back pain patients was significant higher than that in the patients with selective operations for other chief complaints (X2= 16.17. P<0.01); the rates of severe complications in diabetic population was much more than those in nondiabetic one, especially the hypertension (P<0.01) and osteoporosis(P<0.05):for the classification of intervertebral disc degeneration, Grade III in nondiabetic population was much more than that in diabetic one (P<0.05), however, Grade V in diabetic one was significant more than that in nondiabetic one (P<0.05); Modic Type 2 in diabetic one was also significant much more than that in nondiabetic one (P<0.05); there was no significant different in the outcomes of operations between two gourps (P>0.05);2. The constancy of the diabetic rat model inducing by STZ and high glucose and fat diet was good; on X ray, there were stenosis of intervertebral space and instability in the diabetic rats after 4-week; the stenosis of the space was significant less in both 8-and 12-week on X ray (P<0.01); the signal intensity on T2-weighte MRI also had a trend of attenuate in the study group (P<0.05); there was good dependability between histological and radiological evaluation;3. The content of water in study group was less and less, the outcomes were (68.2±6.2)% and (60.1±9.7)% in 8- and 12-week, respectively. There were statistical differences compared with those at the same time points (P<0.05, P<0.01); the content of GAG in study group was also decreased, and there was statistical differences at 8-week compared with control group (P<0.05); the expressions of aggrecan and collagen type II were decreased, but that of collagen type I was increased; when measured by qPCR, the mRNA level of MMP-3 was increased at both 8- (P<0.05) and 12-week (P<0.01); the mRNA level of BMP-2 was increased at 4-week (P<0.05), but decreased after that time point; the mRNA level of TIMP-1 was only increased at 12-week (P<0.05). Conclusion:1. Diabetes mellitus is one of the influencing factors of disc degeneration:there are much more severe complications in diabetic population than that in nondiabetic one; the end-stage of disc degeneration and Modic changes type 2 was much more in diabetic one:there is no difference in the outcomes between two groups;2. The constancy of diabetic rat model inducing by STZ is good, which could be used as an animal model for the studying of diabetes mellitus and disc degeneration; the disc degeneration is progressed when the diabetes mellitus is coexistence; 3. The contents of water and aggrecan were decreased in the intervertebral disc with diabetes mellitus, and the ratio of type I/II was increased; the abnormality changes of metabolic factors including MMP-3, BMP-2 and TIMP-1 may induce the disorders of extracellular matrix and the influencing factor of disc degeneration.更多还原