【作者】 申庆丰；

【导师】 张庆俊；

【作者基本信息】 河北医科大学 ， 外科学， 2004， 硕士

【摘要】 目的：脊髓空洞症是缓慢进展的退行性疾病，迄今为止对其形成机理尚不完全清楚。脊髓空洞前状态是近几年在研究脊髓空洞症发病机制中提出的一个新的概念，由于它反映了脊髓空洞症发病的早期表现，而且可以被逆转，所以对脊髓空洞症的预防和治疗具有重要意义。 我们先前实验显示，脊髓空洞前状态的病理变化主要为缺血水肿样改变。这种水肿为血管源性水肿，主要与血管内皮通透性增加和血脑脊液脊髓屏障的损害有关。基质金属蛋白酶-9(matrix metalloproteinase 9，MMP-9)可以降解Ⅳ型，Ⅴ型胶原，纤维连接蛋白，弹性蛋白和变性后的基质胶原，内皮细胞间的紧密连接以及构成血脑脊液脊髓屏障的主要成分基质层，故它可以破坏血管内皮和血脑脊液脊髓屏障使二者通透性增加，造成血管源性水肿。本实验拟通过观察家兔脊髓空洞症模型的空洞形成前期脊髓的组织学变化，测定脊髓含水量，应用 western blot 和免疫组织化学方法对MMP-9 的表达进行研究，并应用 Doxycycline 进行干预治疗旨在探讨 MMP-9 在脊髓空洞前状态形成中的作用，为临床防治脊髓空洞症提供理论依据。 方法：将 96 只健康、体重 1.5-2kg 的中国白兔，随机分为四组，其中 Kaolin 组 30 只，Doxycycline 治疗组 30 只，生理盐水组 30 只和正常对照组 6 只。家兔在氯胺酮（30mg/kg）耳缘静脉麻醉，氯丙嗪（30mg/kg）肌注辅助──────────────────────────────────────── 1<WP=4>中 文 摘 要麻醉后，取俯卧颏低位，，颈项部手术区常规消毒，按McLaurin 法，用 4 号针头行经皮枕大池穿刺术，缓慢抽出无色透明脑脊液 0.6ml 后，脊髓空洞症模型组（Kaolin 组）和 Doxycycline 治疗组动物注入 37℃25% Kaolin 悬浊液0.6ml；依同样方法生理盐水组仅注入 37℃生理盐水 0.6ml。Doxycycline 组动物给予 Doxycycline 25mg/kg.d 胃管内注入。正常对照组麻醉后处死。在第 1、3、7、10、14 天，各时间点随机抽取 Kaolin 组，Doxycycline 组动物和生理盐水组各 6 只，处死后采集颈髓组织标本。各组动物分别在不同时间分批用氯胺酮 50mg/kg 快速推注，断头处死。将动物脑和颈髓组织迅速取出，于 C1-2 节段截取 4mm，仔细剔除表面蛛网膜及 Kaolin 沉积，滤纸吸除表面水分，立即应用于脊髓含水量的测定，置电子天平（分度值 0.0001g）称取湿重(W)，再经 110℃ 恒温烤箱烘烤 24 小时后称取干重(D)，应用公式：脊髓含水量(%)=(W – D)/ W × 100 ，计算出脊髓含水量；截取C2-3节段标本置4%多聚甲醛固定48小时后，常规脱水，石蜡包埋，制作 5μm 厚连续切片。进行 H&E染色和免疫组织化学染色观察组织病变程度及 MMP-9 表达水平；截取 C3-4节段标本迅速放入液氮中 2 分钟，-70℃冰箱保存，应用 Western blot 测定胞浆 MMP-9 活性。统计学分析应用 SPSS10.0 软件包进行方差分析检验比较各 kaolin组、doycycline 组及生理盐水组和正常对照组间的 MMP-9活性的差异，数据以均数±标准差表示。 结果 ：术后第 1-3 天，Kaolin 组动物表现出精神萎靡，嗜睡，厌食；于 7-14 天症状逐渐加重，且出现肢体无力，轻瘫及运动协调性差等神经功能障碍，并伴有明显消瘦，其中──────────────────────────────────────── 2<WP=5>中 文 摘 要1 只因处于濒死状态而处死。生理盐水组和正常对照组动物在观察期间则表现正常。Doxycycline 组动物也有精神萎靡，嗜睡，厌食等表现但仅有 8 只出现肢体无力和轻瘫。应用干湿法测定观察，生理盐水组各时间点测定的脊髓含水量和正常对照组家兔相比均无明显统计学差异；Kaolin 组各时间点动物脊髓含水量与生理盐水组各时间点和正常对照组比较有明显升高；Doxycycline 组动物各时间点动物脊髓含水量与kaolin 组比较有明显降低。肉眼观察生理盐水组和正常对照组动物脑、脊髓、蛛网膜、蛛网膜下腔、中央管外观正常。Kaolin 组动物的脑脊髓标本在第 1 天肉眼观察变化不明显，仅于下位脑干至上颈髓背、腹侧蛛网膜下腔可见大量 Kaolin沉积，四脑室正中孔和侧孔被不同程度阻塞；第 3 天除上述变化外，开始出现脑组织肿胀、脊髓肿胀增粗；第 7、10、14 天水肿最为明显，枕大池狭窄或闭塞，四脑室轻度扩张，Kaolin 肉芽肿形成，与蛛网膜下腔和脊髓粘连，不易剥离。Doxycycline 组动物的脊髓标本第 1、3、7 天均无明显变化，只见下位脑干至上颈髓背、腹侧蛛网膜下腔有大量 Kaolin 沉积，四脑室正中孔和侧孔被不同程度阻塞；第 10、14 天脊髓标本可见轻度肿胀增粗，较 Kaolin 组肿胀轻微，亦有 kaolin肉芽肿形成，与网膜下腔和脊髓粘连，不易剥离。光镜观察生理盐水组和正常对照组动物脊髓神经元轮廓正常，核及核仁清楚，胞浆内尼氏体分布均匀，神经元、神经胶质细胞、血管周围间隙正常。Kaolin 组动物术后第 1 天，脊髓组织学检查亦无明显改变；第 3 天出现细胞、血管?更多还原

【Abstract】 objective: Syringomyelia is a kind of chronic disease.It’smechanism is still incompletely clear, and for this reason thecause of disease become the focus to dispute. Presyrinx state is anew concept that was put forward in study mechanism ofSyringomyelia in the last few years. because it can indicate theearlier period expression of syringomyelia, research of thepresyrinx state have great use in preventing and treatingsyringomyelia. Our experiment showed: presyrinx state is a kind ofchanges for lack in blood. This kind of swollen is asogenicedema .the main cause is disruption of the blood-brain barrierand abnormal vascular permeability. Matrix metallo-proteinase-9 can degrades gelatin ,collagen Ⅳ, Ⅴ; elastin;virtronectin; myelin basic protein; and other subtrates. as to this,it can disrupt the blood – brain barrier and increase the vascularpermeability. so we think MMP-9 may plays an important rolein presyrinx state. doxycyline is artificially synthized inhabitorof MMP-9. it can inhabit MMP-9 activity. This experimenttarget to investigate the role and mechanism of MMP-9 inthe formation of presyrinx state of experimental syringomyelia──────────────────────────────────────── 6<WP=9>英 文 摘 要in rabbits，and supply the basis to the therapy and prevention tosyringomyelia. We estimate the degree of the spinal cord edemaand observed its development tendency.In the same time , theMMP-9 expression intensity in spinal cord was determined bywesternblot and immunohistochemistry,moreover,we applieddoxycyline to interfere the MMP-9 activity. Method: 96 Chinese white rabbits, weight 1.5-2 kg, isdivided randomly into fuor groups: Kaolin group(30 rabbits),the group treated with doxycycline (30 rabbits), physiologicalsaline group(30 rabbits) and contral group(6 rabbits). Underketamine anesthesia (30 mgs/ kg),0.6ml of 25% kaolin solutionwas injected into the cisterna magna after 0.6ml of cereberalspinal fliud was withdraw slowly from the cistern, in all rabbitsof the kaolin group and doxycycline group,while 0.6ml of 37℃physiological saline was injected into the cistern in those ofsaline group; the doxycycline (25mg/kg.d) after the operationwas applied for doxycycline group. At first, third, seventh,tenth, forteenth day after kaolin injection, the rabbits selectedrandomly from every group were anaethetized again. All theanimals were killed by trasecting at junction of cervico-thorciccord, then the brains and cervico cords were moved wholly andquicly, 4mm cervico cord were cut from C1-2 segement,the wetweight(W) was obtained with electronic balance(0.0001g) afterclearing off carefully the arachnoid, kaolin deposition andwater from surface. The dry weigh(D) was scaled after roastingfor 24 hours in 110℃ thermostatic baker .Water content of──────────────────────────────────────── 7<WP=10>英 文 摘 要spinal cord (%) can be obtained with the formula:watercontent(%)=(W-D)/W×100.The spcimens of C2-3 were fixedin 4% formaldehydum polymeridatum for 48 hours, thenembeded in the paraffin . H﹠E staining and imumuno-histochemistry were completed to the section. Take thesection C3-4 a specimen to put into the liquid nitrogen for 2minuteses quickly,then keep it in -70 ℃ refrigerators. Westernblot measureses the expression of MMP-9 activity. Theexpression of MMP-9 at different time pionts was comparedwith edema level of spinal cord at corresponding time point,while both of the development tendencys were observed. Result: All animals of kaolin group had showndepressd,anorexia from the first to third day after acupuncture,most severely during the seventh to the fourteenth day inaddition to having the slight paralysis in this period; thesesymptoms were remissive after 2 weeks. The animals ofdoxycycline group had shown depressd,anorexia from the fi更多还原