【作者】 赵军；

【导师】 权正学；

【作者基本信息】 重庆医科大学 ， 外科学， 2008， 硕士

【摘要】 随着工农业、建筑及交通事业的发展,脊髓损伤(spinal cord injury,SCI)的发病率有逐渐增加的趋势。SCI所导致的功能损害(如截瘫),不仅给患者造成巨大的身心痛苦,而且还给家庭和社会带来沉重的经济负担和社会问题。目前,SCI研究的进展相对缓慢,预后常难于估计,主要是SCI的病理生理机制非常复杂,对其认识不全。因而建立一个适宜的SCI模型,是进行SCI基础研究的前提。1911年,Allen报道了垂直打击脊髓建立SCI模型的方法,因这种方法更接近于人类SCI过程,临床相关性好,已广泛用于SCI的研究、治疗。现在,大鼠急性SCI打击模型运用较广泛,并为人类SCI的研究做出了巨大贡献。随着对SCI研究的深入,探索某一基因或细胞因子对SCI后的影响变得越来越重要。小鼠不仅成本低、好喂养,而且在基因研究方面比大鼠更具明显优势,其基因与人类有着高同源性,人类对小鼠遗传操作技术也较成熟,便于分析某一基因或细胞因子对SCI后的影响,因此我们建立小鼠急性SCI打击模型并对其进行评价。锌指蛋白A20是近年在细胞内源性保护机制的研究中发现的一类新的蛋白质分子,脂多糖(lipopolysaccharide,LPS)或肿瘤坏死因子(tumor necrosis factor,TNF)等刺激因素均可诱导内源性锌指蛋白A20的快速表达,而A20通过终止LPS或TNF诱导的单核巨噬细胞系统核因子-κB(nuclear factor-kappaB,NF-κB)活化来限制继发性损伤反应是很关键的,对NF-κB的活化具有重要的负反馈调控作用,是防止体内炎症反应失控的重要的内源性调控蛋白。A20研究的兴起,为深入揭示机体内源性保护机制以及进一步抑制创伤感染及其并发症的发生、发展提供了一条新的途径。而SCI性截瘫并发感染时,在内毒素刺激作用下,脊髓组织内锌指蛋白A20的表达变化规律及其与神经元继发性损害的相互关系以及神经元内源性保护机制研究等一系列相关问题,需待进一步研究与探讨。目的:1、建立并评价不同力致伤的小鼠Allen’s脊髓损伤模型,为进一步研究脊髓损伤机制提供实验动物参考。2、初步研究SCI合并感染时小鼠脊髓组织中锌指蛋白A20的表达规律及其对脊髓损伤病理改变的影响。方法:1、将153只小鼠随机分为假手术组(18只)和实验组(135只),实验组按打击重量及高度分为A组(2g×2.5cm,45只),B组(3g×2.5cm,45只),C组(3g×5cm,45只)。假手术组只暴露脊髓,实验组显露脊髓后采用改良Allen法致伤。伤后行运动功能BMS评分、MEP电生理测定及病理切片HE染色。2、选用健康昆明种小白鼠95只,雌雄不拘,体重18～22g,制作SCI及SCI合并感染模型。随机分为对照组、单纯SCI组、单纯内毒素组和SCI内毒素组。行病理组织学观察及BMS评分。结果:1、实验组打击后运动功能损伤明显,但后期都有不同程度恢复,A组2w后BMS评分与对照组无显著性差异(p>0.05);C组恢复最差,死亡率最高;B组死亡率低,恢复不完全。实验组在损伤后3d内均出现MEP-N1峰潜期延长,之后逐渐降低;A组1w后MEP已与对照组无显著差异(p>0.05);B、C组8w时MEP仍显著延长。不同组、不同观察时间点病理切片也有不同改变。2、各时间点单纯SCI组、SCI内毒素组与单纯内毒素组BMS评分差异显著(p<0.05),5d、7d时SCI组、SCI内毒素组BMS评分存在显著性差异(p<0.05)。对照组和单纯内毒素组脊髓均未见明显充血水肿,SCI组术后1d～3d血肿逐渐消退,SCI内毒素组术后5d仍见脊髓部分充血水肿,至7d才逐渐消退。对照组和单纯内毒素组均未见神经元细胞胞质锌指蛋白A20明显阳性表达,SCI组术后8h、12h、1d可见A20阳性表达;SCI内毒素组于手术后2h可见A20阳性表达,一直持续到术后3d。结论:1、致伤力为2g×2.5cm,3g×2.5cm ,3g×5cm时,可复制出小鼠轻、中、重度急性SCI模型,该模型所致的脊髓损伤结果稳定可靠,为SCI的理论研究奠定了基础。2、锌指蛋白A20作为一种参与体内炎症反应调控的内源性蛋白,在SCI及SCI合并感染时,脊髓组织中即可呈现出阳性表达,SCI合并感染时表达时间显著延长。A20是机体的一种重要的内源性抗炎保护效应机制,可能对于调控SCI感染时脊髓适度的炎症反应具有重要意义。更多还原

【Abstract】 As the development of industry, agriculture, architecture and transportation, the incidence of spinal cord injury (SCI) is on the tendency of increasing gradually. The functional lesion such as paraplegina caused by SCI not only brings enormous physically and mentally painful to the patient himself, but also spell heavy economic burden and social problem to the family and society. At present, the investigative procession of SCI is relatively slow that the prognosis is hard to estimate, as the pahophysiological mechanism of SCI is so complicated that we haven’t completely recognize it. So establishing an eligible SCI model is the prerequisite for SCI foundmental investigate. Allen has report the model of vertical coup to the injury in 1911, which was more close to mankind spinal cord injury process and had good clinical dependability. It is widely used in SCI study and treatment. Now, rat acute SCI model is applied extensive, as well as to make tremendous contribution to human SCI therapeutic research. But to accompany with the research of SCI, it becomes more and more important to explore the importance of one certain gene or cytokine related to the prognosis. Mice is not only low cost, easy to feeding, but also has more predominace in genetic research aspect. It’s relatively easy for us to analyze the importance of one certain gene or cykokine to the prognosis, because the mice’s gene has high homology with mankind, and the mice genetic manipulation technique is also mature. So we establish mice acute SCI model and evaluate it.Zinc finger protein A20 is a new group developed recently in cytoendogenous protection mechanism research. The stimulating factor such as LPS or TNF can induce quick expression of endogenous zinc finger protein A20.A20 is the key to limit the secondary lesion reaction by terminating activation of NF-kB which induced by LPS or TNF. It has reverse feedback regulation effect for activation of NK-kB, and is an important endogenous modulin which can prevent an uncontrolled inflammatory reaction in vivo. The discovery of A20 has provided an original pathway to reveal organisim endogenous protection mechanism in advance and inhibited traumatic infection and the occurrence, development of its complication. But when there is SCI paraplegia complicated with endotoxemia, a trail corresponding problem such as the expressive change rule of A20 in myeloid tissue, the interrelationship with neuron secondary lesion and neuron endogenous protection mechanism under stimulatory function of endotoxin is still need of a further study and approach.OBJECTIVE1.To establish and evaluate a mouse model of graded Allen’s spinal cord injury(SCI), and provide theoretical evidence for a new model of contusive SCI.2. To study initially the expression of zinc finger protein A20 of mouse myeloid tissues in the process of spinal cord injury with infection and the effect on the posttraumatic secondary endotoxic spinal cord injury.METHODS1.A total of 153 mice were divided into surgical controls (n=18)that received laminectomy but did not receive a contusive injury, and experimental groups(n=135)ruined by modified Allen’s method and distinguished by the amount of weight in grams dropped onto the impounder and height from the weight to the impounder: group A(2g×2.5cm n=45), group B(3g×2.5cm n=45) and group(3g×5cm n=45).All animals were assessed by electrophysiological, structural and examined using the Basso Mouse Scale (BMS).2.The mode of SCI and SCI with infection were made.Ninty-five healthymouse of both sexes with a mean body weight of 20 g(18～22 g)were randomized into 4 groups:control,SCI,LPS and SCI plus LPS.And tested by pathohistologic observation and BMS score. RESULTSThe BMS revealed a statistically significant difference between surgical controls and experimental groups after injury. In experimental groups, there was no difference in functional outcome between group A and surgical controls after week 2; group C recovered badly with a high mortality and there was no recovery evidence in group B up to week 8. The mean peak latency of N1 wave in MEP prolonged by day 3 and then gradually decreased in experimental groups; however, by week 1 there were no significant difference between group A and surgical controls(p>0.05); in group B and C, there were still significant decrement by week 8. there were different Histopathological changes in groups at tested time.There was significant difference between SCI group and SCI plus LPS group in day 5 and day 7(p<0.05). Both control group and LPS group had no obvious congestion and hydropsia. Hematoma subsided within 3 days in SCI group. There had congestion and edema partly in day 5 in SCI plus LPS, which subsided until day 7.Neither control group nor LPS group had obvious positive expression of A20 in neural cellular kytoplasm.A20 positive expression existed in SCI group in 8h ,12h and day 1;and in SCI plus LPS group in 2h which continue to day 3.CONCLUSIONS1.A mouse model of contusive spinal cord injury is a reasonable model and provides theory basement for SCI studies. 2.Zinc finger protein A20 is highly expressed as an endogenous anti—inflammatory protein which participate the inflammatory reaction in vivo.It expressed positively in SCI alone and SCI with infection in which state its expression significantly prolong. It further indicates that expression of A20 in tissues following SCI with infection might play an important role in prevention of uncontrolled inflammatory response.更多还原