摘要
目的:通过手术破坏纤维环和软骨终板建立自免疫椎间盘炎的动物模型,重点探讨其病变过程中的影像学变化及免疫学表达,深入探讨自免疫因素在椎间盘炎中的作用,使动物模型更具有实际应用价值,进而为临床诊断和治疗的创新提供理论基础。
方法:选择健康成年新西兰大耳白兔50只,雌雄不限,随机分为三组:手术组(30只),经前外侧入路手术破坏腰椎间盘纤维环及上位软骨终板,建立兔椎间盘炎动物模型;设立假手术组(10只)及对照组(10只)。对照组即刻行X线及MRI检查,处死取腰3/4、腰4/5、腰5/6椎间盘标本。假手术组同手术组取相同手术入路,暴露椎间盘但不破坏纤维环和软骨终板。手术组与假手术组分别于术后1周、2周、4周、8周、12周分批处死动物,处死取材前行X线片、MRI检查。取手术区椎间盘标本,行:(1)大体病理切片,观察椎间盘及椎体炎症变化;(2)常规HE染色,形态学比较及淋巴细胞的表达;(3)应用免疫组化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。手术组与假手术组、对照组进行比较以分析各指标的表达及变化过程,探讨自免疫反应在椎间盘炎中的作用;(4)细菌学培养。
结果:1.影像学表现:手术组常规X线检查在术后1周未见异常征象,术后2周显示手术区椎间隙狭窄,术后4周手术椎体骨质疏松透过度增加,术后8周上位椎体密度不均匀,变化累及下位椎体,术后12周可见变化累及整个上位椎体,骨质增生、破坏同时存在。手术组动物术后2周MRI出现上位椎体终板及终板下骨的炎性表现,矢状位T2WI上即可见手术椎间盘信号异常,呈不同程度的破坏、碎裂、变小,正常髓核的高信号消失,呈低信号改变。终板及终板下椎体松质骨有不同程度的侵犯破坏致椎体和椎间盘分界模糊不清,相邻椎体受累,T2WI上呈混杂斑点状高信号。术后4周MRI显示椎间盘信号改变进一步加重,并且手术区下位椎体的终板及终板下骨出现信号改变,术后12周T2WI上可见手术椎间盘相邻上位整个椎体高信号。假手术组与对照组均未见异常。2.大体病理:三节段椎体矢状位切片并常规染色显示手术区椎间盘、终板及终板下骨炎性改变,淋巴细胞浸润。3.HE染色:光镜下可见手术组椎间盘内、外层纤维环紊乱,失去正常结构,胶原纤维不连续、增生,外层纤维环成纤维细胞增多,术后1-4周可见淋巴细胞浸润,近纤维环内层髓核部分有大量软骨细胞增生。假手术组及对照组则无上述表现。4.免疫组化染色:手术组1周、2周、4周的标本可见近边缘细胞浸润区有CD4阳性、CD8阳性淋巴细胞分布,其中CD4阳性淋巴细胞主要在术后1周、2周取材的标本表达较多,CD8阳性淋巴细胞则在术后2周、4周取材的标本表达较多。而假手术组则于术后1周、2周可见有极少量淋巴细胞表达,对照组未见淋巴细胞表达。手术组淋巴细胞表达与假手术组、对照组比较具有统计学意义。5.免疫荧光:手术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周、4周、8周大量表达。假手术组及对照组均可见IgG极少量表达,未见IgM表达。手术组与假手术组、对照组比较具有统计学意义。6.细菌培养:阴性。
结论:1.运用前外侧入路手术破坏纤维环及软骨终板建立椎间盘炎动物模型的方法,其影像学表现符合临床文献对椎间盘炎的报道;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,并可以累及相邻未破坏终板之椎体,且未见细菌化脓性炎症反应;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。
Objective:Establishing discitis animal model by destroying the fibrous ring and the cartilage lamina through the surgery,discussing the mechanism of autoimmune reaction in the discitis by observing its image performance and the changing process of autoimmune condition.
Methods:50 healthy adult New Zealand white rabbits no matter the sexual were enrolled and divided into three groups:The surgery group(30):The rabbit discitis animal model was set up by using the anterior external surgery to destroy the fibrous ring and the cartilage lamina;Sham group(10)and control group(10).Control group were executed to get the intervertebral disc specimens after took the X-ray and the MRI instantly.The surgery group and Sham group were executed on 1 week,2 weeks, 4 weeks,8 weeks,12 weeks after surgery,to get the intervertebral disc in the surgical area after took the X-ray and the MRI.The specimen were used to:(1)Conventional HE stain,morphology comparison and lymphocyte expression;(2)Masson stain, observing the inflammatory fibrosis.(3)Observing the expression of the CD4/CD8 positive lymphocyte and the IgG/IgM by using immunocytochemistry and immumofluorescence.Analysising the expression and the change process of the indexs by comparing the three groups to discuss the role autoimmune reaction plays in the discitis.(4)Bacteriological cultivation.
Results:1.Imaging findings:On 2 weeks after surgery,abnormalities on X-ray imaging can be see in the surgery group.On 4 weeks after surgery,the intervertebral space showed narrow,inflammatory reaction.On 12 weeks,destruction and sclerosis of centrums ossein were observed.On 2 week,abnormal signal on intervertebral disc and different extent of destruction,fragmentation,shrinking was showed in MRI,and on T2WI there was low signal change instead of high signal of normal nucleus pulposus,destruction on cancellous bone of cartilage lamina and Under the cartilage lamina was different,which led to the the boundary between centrum and intervertebral disc became unclear.On 4 week,the neighboring centrum was also affected,on T2WI assumed the combination spot shape of T2 high signal.2.HE stain: Under the light microscope,interior/external layer fibrous ring showed confused and lost the normal structure in surgery group.Fibroblast in external layer fibrous ring increased;Hyperplasia of cartilage cell occurred in the nucleus pulposus next to the interior layer fibrous ring and the nucleus pulposus became thicker.But there were no the same change in Sham group and control group.3.Masson stain:Under the light microscope,the color of the external layer fibrous ring was green in surgery group,the surrounding muscular tissue showed red,inflammation reaction led to muscle fibrosis and the area between the external layer fibrous ring and the muscle showed gray.4. Immunohistochemistry stain:on 1 weeks,2 weeks,4 week after the surgery,the CD4/CD8 positive lymphocyte can be seen in the area next to the surgical.The CD4 positive lymphocyte mainly expressed on 1 week,2 weeks,the CD8 positive lymphocyte mainly expressed on 2 weeks,4weeks.But only little expression of lymphocyte assumed on 1 week,2 weeks in sham group,no expression of lymphocyte can be seen in control group.5.Immumofluorescence:in surgery group, we can observe little expression of IgG,IgM on 1 week after the surgery and massive expressions on 2 weeks,4 weeks,8 weeks.Little expression of IgG showed in the sham group and the control group.6.The result of bacteriological cultivation was negative.
Conclusions:1.Discitis animal model can be established by using the anterior external surgery to destroy the fibrous ring and the cartilage lamina.Its imaging consistent with the representation reported in the clinic literature.2.Contact between the autoantigen of the nucleus pulposus and the blood circulation will provocate autoimmune reaction after destroying the fibrous ring and the cartilage lamina,the autoimmune reaction can be display in both cellular immunity and humoral immunity; 3.Autoimmune reaction plays a vital role in pathogenesy,pathological change and symptom generating of discitis.
引文
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