异常力学负荷导致椎间盘退变的实验研究  

Experimental Study of Lumbar Intervertebral Disc Degeneration Caused by Abnormal Mechanical Load

作　　者：毛强[1,2] 何帮剑 张圣扬[1,2,3] 肖鲁伟 童培建[1,2] 华江[1,2] MAO Qiang;HE Bangjian;ZHANG Shengyang;XIAO Luwei;TONG Peijian;HUA Jiang(Department of Orthopedics and Traumatology,The First Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou 310053,China;Department of Orthopedics and Traumatology,Zhejiang Provincial Hospital of Chinese Medicine,Hangzhou 310053,China;The First Clinical Medical College of Zhejiang Chinese Medical University,Hangzhou 310053,China)

机构地区：[1]浙江中医药大学附属第一医院骨伤科,杭州310053 [2]浙江省中医院骨伤科 [3]浙江中医药大学第一临床医学院

出　　处：《中国中医骨伤科杂志》2020年第12期1-6,共6页Chinese Journal of Traditional Medical Traumatology & Orthopedics

基　　金：浙江省自然科学基金(LQ16H270007);国家自然科学基金(81603639);浙江省中医药科学研究基金项目(2020ZA050)。

摘　　要：目的:观察异常力学负荷是否通过激活IL-1β/Wnt-β-catenin信号通路导致椎间盘退变。方法:选用SPF级雌性C57BL/6小鼠12只,8周龄,体质量20~30 g,随机分为正常对照组和椎间盘退变模型组,每组6只。诱导椎间盘退变模型组小鼠保持长时间的后足站立姿势,增加小鼠腰椎的轴向力学负荷,每天共5 h,维持8周;空白对照组小鼠不加任何干预。取各组小鼠腰椎间盘组织进行苏木精-伊红(HE)染色观察其病理学改变;免疫组织化学染色和Western Blot检测其Ⅱ型胶原(Col2a1)、蛋白聚糖(Aggrecan),IL-1β,β-catenin、基质金属蛋白酶-3和基质金属蛋白酶-13(MMP-3和MMP-13)等蛋白的表达;qRT-PCR检测Col2a1,Aggrecan,IL-1β,β-catenin,MMP-3和MMP-13等基因的表达。结果:与正常对照组小鼠比较,椎间盘退变模型组小鼠的腰椎间盘组织HE染色结果显示椎间盘细胞数量和细胞外基质含量减少,纤维环结构紊乱,软骨终板厚度降低;免疫组织化学染色和Western Blot检测结果显示Col2a1和Aggrecan蛋白表达降低(P<0.01),而IL-1β,β-catenin,MMP-3和MMP-13的活性增强(P<0.01);qRT-PCR检测结果提示Col2a1和Aggrecan基因的表达显著下降(P<0.01),而IL-1β,β-catenin,MMP-3和MMP-13基因的表达明显上调(P<0.01)。结论:异常力学负荷能引起小鼠椎间盘出现退变的组织病理学表现,导致椎间盘细胞外基质的组成成分减少,MMPs的活性增加,符合椎间盘退变的主要特征。说明该方法能成功构建腰椎间盘退变小鼠模型,是一种有效模拟人类椎间盘退变的致病因素且简便易行的椎间盘退变小鼠模型制备方法;异常力学负荷可能通过激活IL-1β/Wnt-β-catenin信号通路导致椎间盘退变的发生发展。Objective:To explore whether abnormal mechanical load leads to lumbar intervertebral disc degeneration by activating the IL-1β/Wnt-β-catenin signal pathway.Methods:12 SPF female C57BL/6 mice(aged 8 weeks with 20~30 g) were randomly divided into the experimental group and the control group(6 mice in each group).Mice in the experimental group were maintained a standing postural position, which increased the axial mechanical load of the lumbar vertebrae for 5 h a day.Mice in the experimental group were performed nothing.After 8 weeks, the lumbar intervertebral disc tissues from two groups were fetched out.The pathological change of the lumbar intervertebral disc tissues were observed by HE staining.The expression of Col2 a1, Aggrecan, IL-1β, β-catenin, MMP-3 and MMP-13 proteins were detected by immunohistochemical staining and Western Blot, and the activity of Col2a1, Aggrecan, IL-1β, β-catenin, MMP-3 and MMP-13 genes was evaluated by qRT-PCR.Results:Compared with the control group, the HE staining results in the experimental group showed that the number of cells and the amount of extracellular matrix were reduced, the fibrous ring structure was disordered, and the thickness of cartilage endplate were diminished.The immunohistochemistry results showed that the expressions of Col2a1 and aggrecan in the experimental group were decreased, whereas the expressions of IL-1β, β-catenin, MMP-3 and MMP-13 were increased as compared with that of control group.The activity of Col2a1 and aggrecan genes in the experimental group was down-regulated compared with the control group, whereas the activity of IL-1β, β-catenin, MMP-3 and MMP-13 genes were up-regulated.Conclusion:Maintaining a standing postural position for a long time can increase the axial mechanical load of the lumbar vertebrae to cause the typical degeneration of lumbar intervertebral disc in mice, which induces decline of the number of cells and upgradation of MMPs extracellular matrix activity.These suggest that this is an simple method to successfully b

关 键 词：椎间盘退变 异常力学负荷 信号通路 

分 类 号：R-33[医药卫生]