自免疫椎间盘炎动物模型构建及其免疫反应

Construction and immune reaction of animal self-immunity discitis models

背景：椎间盘炎概念的最初含义是化脓性感染。而实践中越来越多人开始关注其自身免疫反应因素。目的：通过手术破坏纤维环和软骨终板建立椎间盘炎的动物模型,探讨自免疫状态表达变化过程。设计、时间及地点：随机对照动物实验,于2007-06/12在天津医科大学总医院骨科生物力学实验室完成。材料：选择健康成年新西兰大耳白兔50只,雌雄不限,随机分为3组：模型组30只;设立假模型组10只及对照组10只。方法：随机取30只兔麻醉后,暴露L3~4,L4~5,L5~6椎间盘,以自制鹅头穿刺针刺入椎间盘,破坏纤维环和软骨终板建立兔腰椎间盘炎模型。假模型组同模型组取相同手术入路,暴露椎间盘但不破坏纤维环和软骨终板。模型组与假模型组分别于术后1,2,4,8,12周分批处死动物,取手术区椎间盘标本。主要观察指标：①常规苏木精-伊红染色,形态学比较及淋巴细胞的表达。②应用免疫组织化学技术和免疫荧光技术观察标本CD4阳性、CD8阳性T淋巴细胞,免疫球蛋白IgG、IgM的表达。③细菌学培养。结果：光镜下可见模型组椎间盘内、外层纤维环紊乱,失去正常结构,炎症反应以及纤维化。假模型组及对照组则无上述表现。免疫组织化学染色显示模型组CD4阳性淋巴细胞在术后1,2周取材的标本表达较多,CD8阳性淋巴细胞则在术后2,4周取材的标本表达较多。而假模型组则极少量淋巴细胞表达,对照组未见淋巴细胞表达。免疫荧光显示模型组可见手术椎间盘近边缘区免疫球蛋白IgG、IgM呈阳性反应,于1周少量表达,术后2,4,8周大量表达。假模型组及对照组均可见IgG极少量表达,未见IgM表达。细菌培养为阴性。结论：①运用前外侧入路手术破坏纤维环及软骨终板可以建立椎间盘炎动物模型。②纤维环及软骨终板受破坏后,髓核等自身抗原可与血液循环接触引发机体自身免疫反应,可表现在细胞免疫和体液免疫两方面。③自免疫反应在椎间盘炎的发病机制和病理演变过程以及症状产生过程中起重要作用。

BACKGROUND： Discitis is an inflammatory lesion of the intervertebral disc. However, it attracts more attention on autoimmune factors. OBJECTIVE： To construct discitis animal model by destroying the fibrous ring and the cartilage lamina through the surgery, in addition, to explore the changes of autoimmune reaction. DESIGN, TIME AND SETTING： Randomized controlled animal experiment was performed at the Orthpeadic Laboratory of General Hospital of Tianjin Medical University from June to December 2007. MATERIALS： Fifty healthy, adult, New Zealand rabbits, irrespective of gender, were randomly divided into the model （n=30）, sham operation （n=10） and control （n=10） groups. METHODS： Thirty rabbits were randomly selected after anesthetized, L3-4, L4-5, L5-6 intervertebral discs were exposed, discitis models were created by destroying the fibrous ring and the cartilage lamina with self-made transfixion pin. Animals in the sham operation group were exposed intervertebral discs without damage. Rabbits in the model and sham operation were executed to get the intervertebral disc specimens at weeks 1,2, 4, 8, and 12 after operation. MAIN OUTCOME MEASURES： （1）Hematoxylin-eosin staining, immunocytochemical comparison and lymphocytic expression. （2）The positive CD4 cells, CD8 positive T cells, as well as the expression of IgG, IgM were detected by immunohistochemistry and immunofluorescence technique. （3）Bacteriological cultivation. RESULTS： Under light microscope, the internal and external layer fibrous rings could be seen disordered, with inflammatory reaction and fibrosis in the model group. However, there was no same appearance in the sham operation and control group. Immunohistochemistry showed that there was more CD4 positive lymphocyte expressed at weeks 1 and 2 after operation, and the CD8 positive lymphocyte expressed more at 2 and 4 after operation. There was a few lymphocyte expressions in the sham operation group, and no expressed in the control group. Immunofluorescence demonstrated that immune globulin IgG, IgM was positive, a little expression was found in the model group at 1 week, which increased at the 2, 4, and 8 weeks after operation. Only a little of IgG expression could be found in the sham operation and control groups. The bacteriological cultivation was negative. CONCLUSION： （1）Discitis animal model can be established by using the anterior external surgery to destroy the fibrous ring and the cartilage lamina. （2）After destroying the fibrous ring and the cartilage lamina, the contact between the autoantigen of the nucleus pulposus and blood circulation will provocate autoimmune reaction, which display in both cellular immunity and humeral immunity. （3）Autoimmune reaction plays a vital role in pathogenesy, pathological change and symptom generating of discitis.