持续压力对离体培养兔脊柱运动节段终板内血管内皮生长因子及β-catenin的影响

Effect of Continuous Pressure on Vascular Endothelial Growth Factor and β-catenin in Rabbit Intervertebral Disc of Motor Segments Endplate in Vitro Culture

目的:研究持续压力对离体培养兔脊柱运动节段终板内血管内皮生长因子(VEGF)及β-catenin表达的影响,进一步探讨VEGF与椎间盘退变之间的分子作用机制。方法:将16只新西兰白兔处死后在无菌条件下取出脊柱运动节段,随机分为对照组与压力组,均放入离体加载和培养装置中培养,其中压力组予持续29.4N压力,对照组不予压力,于培养前及培养后第3,7及14天,两组各取10个样本,检测椎体终板内血管芽数量,应用免疫组化与Western blot检测VEGF及β-catenin的表达情况。结果:在持续压力条件下椎体终板形态逐渐被破坏,血管芽数量显著下降,与对照组比较差异有统计学意义(P<0.05);免疫组化检测,持续压力下培养1周后,终板内VEGF染色强度显著下降(0.182±0.063),与对照组(0.237±0.051)相比差异有统计学意义(P<0.05),培养2周时染色强度进一步降低(0.156±0.035),差异有统计学意义(P<0.05);相反,持续压力上调了终板内β-catenin的表达,培养期间与对照组相比差异均有统计学意义(P<0.05);Western blot结果显示,与新鲜标本及对照组比较,持续压力同样引起VEGF显著下降,差异有统计学意义(P<0.05),却上调了β-catenin的表达。结论:持续压力导致离体培养兔脊柱运动节段内椎体终板血管芽数量减少、VEGF表达下降、β-catenin表达上调,提示持续压力可能是通过Wnt/β-Catenin信号通路调节椎体终板内VEGF表达。

Objective：To study the effect of continuous pressure on the expression of vascular endothelial growth factor（VEGF）and β-catenin in vertebral endplate,and further explore the molecular mechanism between VEGF and intervertebral disc degeneration.Methods：Sixty New Zealand rabbits were sacrificed after removed under sterile conditions in the spinal motion segment,were randomly divided into control group and pressure groups were placed in vitro loading and culture device for culture.In the pressure group,which were treated with consistence pressure 29.4 N,the control group did not pressure.At the 3rd,7th,14th day after culture and pro-culture,10 samples were collected from each group,and the number of vertebral endplate vascular bud and the expression of VEGF and β-catenin were detected by immunohistochemistry（IHC）and Western blot.Results：The morphology of the vertebral endplate was gradually destroyed,and the number of vascular buds decreased significantly under the consistence pressure condition,and there was a statistical difference compared with the controls（P〈0.05）.IHC staining showed that the VEGF staining intensity in the endplate decreased significantly（0.182±0.063）after 1 weeks of culture under pressure.Compared with the control group（0.237±0.051）,the staining intensity was significantly different（P〈0.05）,and the staining intensity was further decreased at 2 weeks（0.156±0.035,P〈0.05）.On the contrary,the consistence pressure increased the expression of β-catenin in the endplate,and the difference was significant compared with that of the control group（P〈0.05）.Western blot results showed that compared with fresh specimen and control group,consistence pressure also caused a significant decrease in VEGF（P〈0.05）but increased the expression of β-catenin.Conclusion：Continuous pressure leads to a decrease of the vascular bud number,a decrease of VEGF expression and an increase of the expression ofβ-catenin in the vertebral endplate of spinal motor segment,which suggests that continuous pressure may regulate the expression of VEGF in the vertebral endplate through the Wnt/β-catenin signaling pathway.