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目的:探讨应用N端30kDa纤连蛋白片段(Fn-f)建立模拟人类椎间盘退变规律的椎间盘退变动物模型的可行性,为椎间盘退变的防治提供实验模型及实验依据。方法:选取雄性6月龄新西兰大白兔28只,麻醉后使用30G微量注射针和微量注射器,在透视引导下经皮将25μl 1.5μmol/L Fn-f(Fn-f组)或25μl磷酸缓冲液(PBS,0.01mol/L,pH值7.2;PBS组)随机分别注射入不同节段的腰椎间盘中心区。分别于注射4、8、12、16周后获取椎间盘,对椎间盘进行组织学检测(HE、Masson三色及番红O染色),并以RT-PCR法对椎间盘聚集蛋白聚糖和Ⅱ型胶原的基因表达水平进行分析。结果:与注射PBS椎间盘相比,注射Fn-f椎间盘造模后4周时椎间盘的髓核、纤维环结构以及胞外基质蛋白聚糖等无明显差别;8周时髓核细胞数量减少、细胞簇状分布,被胞外基质分隔开来,纤维环层状结构排列部分出现紊乱,蛋白聚糖染色变浅;12和16周时髓核细胞数量明显减少,细胞变圆,呈明显的成簇聚集分布,纤维环排列明显不规整,各层间出现裂隙,甚至断裂,蛋白聚糖染色明显变浅,甚至部分未见染色。Fn-f组椎间盘聚集蛋白聚糖mRNA表达水平在8、12和16周3个时间点均明显低于PBS组(P<0.05);Ⅱ型胶原mRNA表达水平在12和16周时明显低于PBS对照组(P<0.05)。结论:透视引导下兔椎间盘内注射N端30kDa Fn-f可诱导椎间盘产生渐进性退行性病变,该法建立的动物模型可作为研究椎间盘退变的发病机制及防治的实验模板。
OBJECTIVE: To investigate the feasibility of establishing an animal model of degenerative disc degeneration using 30kDa fibronectin fragment (Fn-f) and to provide a theoretical and experimental basis for the prevention and treatment of disc degeneration. Methods: Twenty-eight male New Zealand white rabbits aged 6 months were anesthetized and perfused with 25μl 1.5μmol / L Fn-f (Fn-f group) or 25μl phosphate buffer (PBS, 0.01mol / L, pH 7.2; PBS group) were randomly divided into different segments of the lumbar intervertebral disc center. The discs were harvested at 4, 8, 12 and 16 weeks after injection, respectively. The intervertebral discs were examined by histology (HE, Masson trichrome and Safranin O staining). The expression of intervertebral disc aggrecan and type Ⅱ collagen The level of gene expression was analyzed. RESULTS: There was no significant difference in the nucleus pulposus, annulus fibrosus and extracellular matrix proteoglycan in the intervertebral discs at 4 weeks after injection of Fn-f disc compared with PBS disc injection. The number of nucleus pulposus cells decreased at 8 weeks, Clustered distribution, separated by the extracellular matrix, the arrangement of fibrous ring layered structure part of the disorder, proteoglycan staining lighter; 12 and 16 weeks, the number of nucleus pulposus was significantly reduced, rounded cells, was significantly Cluster clustered distribution, annulus fibrosus arranged significantly irregular, cracks appear between the layers, or even broken, proteoglycan staining was significantly lighter, and even part of no staining. The mRNA expression of intervertebral disc aggrecan mRNA in Fn-f group was significantly lower than that in PBS group at 8, 12 and 16 weeks (P <0.05). The mRNA expression of type II collagen was significantly lower at 12 and 16 weeks PBS control group (P <0.05). CONCLUSION: Fluorocarbon-mediated 30kDa Fn-f injection at the N-terminal of the intervertebral disc can induce progressive degeneration of the intervertebral disc. The animal model established by this method can be used as an experimental template for studying the pathogenesis and prevention of disc degeneration.