摘要
目的探讨血管内皮生长因子(VEGF)/血管内皮生长因子受体2(VEGFR2)信号轴激活促进腰椎间盘退变的分子机制。方法通过经腹膜外入路手术方法在小鼠体内建立腰椎间盘退变模型,采用组织学染色评估建模后腰椎间盘退变情况,采用免疫荧光染色检测退变腰椎间盘内VEGF、VEGFR2、基质金属蛋白酶(MMP)-13等指标的表达;另外通过体外实验对髓核细胞分别进行VEGF干预及小干扰RNA(siRNA)沉默VEGFR2,采用实时定量反转录聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)分析VEGF/VEGFR2通路激活与MMP-13表达增加之间的关系。两组间比较应用独立样本t检验,多组间比较采取单因素方差分析。结果本实验通过在小鼠体内构建腰椎间盘退变模型,建模后腰椎间盘内出现典型的腰椎间盘退变病理特征,组织学评分高于假手术组[组织学评分(11.17±0.44)分比(4.33±0.17)分,t=14.50,P<0.01],建模成功。免疫荧光染色发现小鼠退变腰椎间盘内VEGF、VEGFR2、MMP-13等指标的表达均高于假手术组;另外通过髓核细胞体外实验发现,加入VEGF干预后MMP-13在RNA水平(F=168.8,P<0.01)及蛋白水平(F=162.5,P<0.01)表达均高于对照组,呈浓度依赖性;而对VEGFR2进行siRNA沉默后,VEGF诱导升高的MMP-13在RNA水平(F=270.9,P<0.01)及蛋白水平(F=131.1,P<0.01)均低于VEGF干预组。结论VEGF/VEGFR2信号轴激活通过促进MMP-13分泌引起腰椎间盘退变。
Objective To identify the role and mechanism of vascular endothelial growth factor(VEGF)/vascular endothelial growth factor receptor 2(VEGFR2)axis activation in degenerative disc disease(DDD).Methods A mouse DDD model was established by anterior stabbing in lumbar intervertebral discs in wild type(WT)mice.After 12 weeks,the mice were sacrificed and the lumbar spine was harvested,embedded in paraffin and sectioned.Safranin-O and fast green staining was applied for histological analysis.Immunofluorescent staining was used to access the expression of VEGF,VEGFR2 and matrix metalloproteinase(MMP)-13 in the discs.In addition,primary bovine disc cells were extracted and stimulated with exogenous VEGF.After Silencing VEGFR2 by small interfering RNA(siRNA),the relation between VEGF/VEGFR2 axis activation and MMP-13 expression in disc degeneration was clarified with real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)and Westen blotting assay.Results Significant disc degeneration was presented in the established mouse DDD model identified by Safranin-O fast green staining(Histological scoring 11.17±0.44 vs.4.33±0.17,t=14.50,P<0.01).Significant elevated expression of VEGF,VEGFR2 and MMP-13 in the degenerative discs was detected by immunofluorescent staining.In vitro study with bovine disc cells showed that the addition of VEGF can promote MMP-13 expression in a dose-dependent way both in RNA level(F=168.8,P<0.01)and protein level(F=162.5,P<0.01).After silencing VEGFR2 by si-RNA,the VEGF-induced elevated expression of MMP-13 was significantly suppressed both in the transcription(F=270.9,P<0.01)and translation level(F=131.1,P<0.01).Conclusion VEGF/VEGFR2 axis activation can promote disc degeneration by promoting the expression of MMP-13.
作者
刘振华
陈翔
李建君
段扬
邱素均
Liu Zhenhua;Chen Xiang;Li Jianjun;Duan Yang;Qiu Sujun(Department of Spinal Surgery,Zhujiang Hospital of Southern Medical University,Guangzhou 510282,China;Department of Pharmacology,the Sixth Affiliated Hospital of South China University of Technology,Foshan 528500,China)
出处
《中华实验外科杂志》
CAS
北大核心
2022年第10期1960-1963,共4页
Chinese Journal of Experimental Surgery
基金
广东省自然科学基金(2021A1515012152)。
关键词
下腰痛
椎间盘退变
血管内皮生长因子
基质金属蛋白酶
Low back pain
Degenerative disc disease
Vascular endothelial growth factor/vascular endothelial growth factor receptor 2
Matrix metalloproteinase
