应用纤连蛋白片段建立椎间盘退变动物模型

Experimental intervertebral disc degeneration induced by fibronection fragment in rabbit

中文关键词:  椎间盘退变  动物模型  纤连蛋白片段  大白兔

英文关键词:Intervertebral disc degeneration  Animial model  Fibronection fragment  White rabbits

中文摘要:

  【摘要】 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。

英文摘要:

  【Abstract】 Objectives: To investigate the feasibility of establishing an experimental animal model by using N-terminal fibronection fragment(Fn-f) which induces the pathologic hallmarks of human intervertebral disc degeneration, and to facilitate the investigations of intervertebral disc degeneration. Methods: 25μl of 1.5μmol/L Fn-f or phosphate buffered saline(PBS) was injected into the central region of isolated lumbar discs in 28 male New Zealand white rabbits of six-month-old under a percutaneous X-guided by using a microsyringe with a 30-gauge needle. Euthanasia was performed at 4-, 8-, 12-, and 16-week, and the histology and gene expression of disc tissue was tested. Results: Histology studies revealed a progressive loss of the normal architecture of the nucleus pulposus(NP) and annulus fibrosus(NF) after injecting Fn-f during 16 weeks compared with PBS injected discs. The pathological sections of the NP region in Fn-f-treated discs at the 4th week showed plenty of nuclear cells clumped loosely with uniform distribution. And rich extracellular matrix was evidenced. At the 8th week, the number of nuclear cells decreased and clustered. At the 12th and 16th week, the Fn-f injected discs showed decreased nuclear cells number and proteoglycan matrix. The NP cells became rounded and clustered. The AF region of the Fn-f-treated discs demonstrated progressive loss of the normal lamellar organization and breakage. Significantly down-regulated expression of aggrecan gene in the Fn-f-treated discs was evidenced at the 8th week, 12th week and 16th week, and the expression of type-Ⅱcollagen gene in Fn-f-treated discs was significantly down-regulated at the 12th week and 16th week compared with PBS treated control discs. Conclusions: 30 kDa of N-terminal Fn-f injection into the central region of rabbit discs results in slowly progressive intervertebral disc degeneration, which is similar to that observed in human disc degeneration and can be used for further study.