压力对离体培养兔椎体终板内血管芽及相关细胞因子的影响
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摘要
目的:研究持续压力对椎体终板内血管及VEGF与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进行培养,其中压力组予持续3kg压力,对照组不予压力,于培养前及培养后第3、7、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:与对照组比较,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(P<0.05);免疫组化检测,持续压力下培养1周后,与对照组同期比较,终板内VEGF染色强度显著下降(P<0.05),培养2周时染色强度进一步降低(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均差异显著(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(P<0.05),却上调了β-catenin的表达(P<0.05)。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,提示持续压力可能通过Wnt/β-catenin信号通路调节椎体终板内VEGF表达。
Objective: To explore the effects of continuous pressure on the expression of vascular and vascular endothelial growth factor(VEGF) and β-catenin in vertebral endplate, and to further explore the molecular mechanism between vascular endothelial growth factor and intervertebral disc degeneration. Methods: Sixteen New Zealand white rabbits were sacrificed and their spinal motor segments were removed under aseptic conditions. They were randomly divided into control group and pressure group. They were cultured in vitro loading and culture device. The pressure group was subjected to continuous pressure of 3 kg, while the control group was not subjected to pressure. Ten samples were taken from each group before culture and3, 7 and 14 days after culture. The number of vascular buds in vertebral endplate was measured and immunized. The expression of vascular endothelial growth factor and β-catenin were detected by immunohistochemistry(IHC) and Western Blot. Results:Compared with the control group, the morphology of vertebral endplate was gradually destroyed and the number of vascular buds decreased significantly under continuous pressure(P<0.05); Immunohistochemical staining showed that the staining intensity of VEGF in endplate decreased significantly after 1 week of continuous pressure culture(P<0.05), and further decreased after 2 weeks of culture(P<0.05); on the contrary, the staining intensity increased after 2 weeks of culture(P<0.05). The expression ofβ-catenin was significantly different from that of the control group during culture(P<0.05). Western Blot results showed that the expression of VEGF was significantly decreased(P<0.05) but the expression of β-catenin was increased by continuous pressure compared with fresh specimens and control group(P<0.05). Conclusion: Persistent pressure resulted in a decrease in the number of vascular buds, a decrease in the expression of VEGF and an increase in the expression of β-catenin in the vertebral endplate of rabbits cultured in vitro, suggesting that persistent pressure may regulate the expression of VEGF in the vertebral endplate via the Wnt/β-Catenin signaling pathway.
Objective: To explore the effects of continuous pressure on the expression of vascular and vascular endothelial growth factor(VEGF) and β-catenin in vertebral endplate, and to further explore the molecular mechanism between vascular endothelial growth factor and intervertebral disc degeneration. Methods: Sixteen New Zealand white rabbits were sacrificed and their spinal motor segments were removed under aseptic conditions. They were randomly divided into control group and pressure group. They were cultured in vitro loading and culture device. The pressure group was subjected to continuous pressure of 3 kg, while the control group was not subjected to pressure. Ten samples were taken from each group before culture and3, 7 and 14 days after culture. The number of vascular buds in vertebral endplate was measured and immunized. The expression of vascular endothelial growth factor and β-catenin were detected by immunohistochemistry(IHC) and Western Blot. Results:Compared with the control group, the morphology of vertebral endplate was gradually destroyed and the number of vascular buds decreased significantly under continuous pressure(P<0.05); Immunohistochemical staining showed that the staining intensity of VEGF in endplate decreased significantly after 1 week of continuous pressure culture(P<0.05), and further decreased after 2 weeks of culture(P<0.05); on the contrary, the staining intensity increased after 2 weeks of culture(P<0.05). The expression ofβ-catenin was significantly different from that of the control group during culture(P<0.05). Western Blot results showed that the expression of VEGF was significantly decreased(P<0.05) but the expression of β-catenin was increased by continuous pressure compared with fresh specimens and control group(P<0.05). Conclusion: Persistent pressure resulted in a decrease in the number of vascular buds, a decrease in the expression of VEGF and an increase in the expression of β-catenin in the vertebral endplate of rabbits cultured in vitro, suggesting that persistent pressure may regulate the expression of VEGF in the vertebral endplate via the Wnt/β-Catenin signaling pathway.
引文
[1]曹旺烽,陈长贤.椎间盘软骨终板退变的研究进展.中国中医骨伤科杂志,2016,24(10):70-74
[2]冯璐,狄国虎,周庆军.血管内皮生长因子及其受体家族在角膜组织及角膜病变中的作用和研究进展.中国组织工程研究,2016,20(11):1644-1650
[3]唐乾利,郭满,吴标良.血管内皮生长因子的研究现状与进展.中国烧伤创疡杂志,2017,29(2):77-87
[4]朱立国,张平,宋庆慧,等.补肾活血方对沙鼠增龄过程中腰椎软骨终板钙化干预作用的初步研究.中国骨伤,2017,11(10):926-932
[5]刘宣,王炎,李丹光,等.丹参酮ⅡA对COX-2激活Wnt/β-catenin信号通路介导的人肠癌细胞VEGF表达的调控作用.中华中医药杂志,2013,28(1):108-112
[6]D Han,C Cao,Y Su,et al.Ginkgo biloba exocarp extracts inhibits angiogenesis and its effects on Wnt/β-catenin-VEGF signaling pathway in Lewis lung cancer.Journal of Ethnopharmacology,2016,192:406-412
[7]J-W Zhan,M-S Feng,L-G Zhu,et al.Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in an Organ Culture.BioMed Research International,2016:1-10
[8]M V Risbud,M W Izzo,C S Adams,et al.An organ culture system for the study of the nucleus pulposus:Description of the system and evaluation of the cells.Spine(Phila Pa 1976),2003,28(24):2658-2659
[9]芮泽,张宁,胡志毅,等.持续性压力促进成骨细胞分泌VEGF的机制.南京医科大学学报(自然科学版),2011,7(7):1002-1006
[10]孙旗,柳根哲,贾育松,等.不同静水压对兔椎体终板软骨细胞相关基因表达的影响.中国矫形外科杂志,2014,22(8):753-757
[11]张小海,徐宏光,吴天亮.椎间盘退变与生物力学.国际骨科学杂志,2010,31(3):137-138,161
[12]彭宝淦,贾连顺,施杞,等.软骨终板钙化在椎间盘退变过程中的作用机理.中国矫形外科杂志,2000,7(2):40-43
[13]王弘,陈学武,徐宏光,等.颈椎病终板软骨组织中血管内皮生长因子和缺氧诱导因子-1α表达的相关性.实用医学杂志,2008,24(16):2745-2747
[14]沈颉.血管内皮生长因子(VEGF)在血管外膜成纤维细胞的表达研究.当代医学,2012,18(24):24-26
[15]王弘,陈学武,徐宏光,等.血管内皮生长因子及其受体在颈椎病椎体软骨终板中的表达变化.实用医学杂志,2008,24(8):1336-1338
[16]王拥军,施杞,周重建,等.益气化瘀方对大鼠颈椎间盘软骨终板内血管的影响.中国中医骨伤科杂志,2002,10(4):3-6
[17]Z Li-Guo,F Min-Shan,Z Jia-Wen,et al.Effect of static load on the nucleus pulposus of rabbit intervertebral disc motion segment in ex vivo organ culture.Chinese Medical Journal,2016,129(19):2338-2346
[18]展嘉文,朱立国,冯敏山,等.丹参注射液含药血清对离体培养兔脊柱运动节段椎间盘的影响.中华中医药杂志,2015,30(5):1834-1837
[19]梁广胜,殷明,刘玉亮,等.Wnt信号通路与椎间盘的发展.中国矫形外科杂志,2014,22(5):441-444
[20]任东成,丁金勇.终板软骨细胞退变相关信号通路的研究进展.中国脊柱脊髓杂志,2017,27(7):657-662
[21]陈志刚,吕书军,张建平,等.β连环素在腰椎间盘退变组织中的表达及意义.江苏医药,2015,41(4):408-410
[22]廖军,谢巧瑜,张乐,等.电针对颈椎病模型大鼠椎间盘纤维环细胞Wnt-β-catenin信号通路的影响.中国针灸,2014,34(12):1203-1207
[23]冯敏山,展嘉文,朱立国,等.体外培养条件下兔脊柱运动节段髓核组织的变化.中国组织工程研究,2015,19(51):8241-8246
[24]朱立国,展嘉文,冯敏山,等.兔椎间盘器官与脊柱运动节段离体培养条件下髓核组织的变化.中国骨伤,2015,28(9):824-831
[25]V Easwaran,H L Sang,L Inge,et al.β-Catenin Regulates Vascular Endothelial Growth Factor Expression in Colon Cancer.Cancer Research,2003,63(12):3145
[26]程华,孙芳玲,艾厚喜,等.Wnt信号通路与血管发生研究进展.神经药理学报,2011,1(6):60-63
[27]江华基,江小成,贺飞林,等.黄芩素通过WNT/β-catenin信号通路促进大鼠腱骨愈合.分子影像学杂志,2016,39(3):297-301
[28]余文珍,陈实,张永亮,等.活血化瘀法对鼠颈动脉粥样硬化损伤血管wnt/β-catenin信号通路的影响.世界中医药,2016,11(3):498-501
[2]冯璐,狄国虎,周庆军.血管内皮生长因子及其受体家族在角膜组织及角膜病变中的作用和研究进展.中国组织工程研究,2016,20(11):1644-1650
[3]唐乾利,郭满,吴标良.血管内皮生长因子的研究现状与进展.中国烧伤创疡杂志,2017,29(2):77-87
[4]朱立国,张平,宋庆慧,等.补肾活血方对沙鼠增龄过程中腰椎软骨终板钙化干预作用的初步研究.中国骨伤,2017,11(10):926-932
[5]刘宣,王炎,李丹光,等.丹参酮ⅡA对COX-2激活Wnt/β-catenin信号通路介导的人肠癌细胞VEGF表达的调控作用.中华中医药杂志,2013,28(1):108-112
[6]D Han,C Cao,Y Su,et al.Ginkgo biloba exocarp extracts inhibits angiogenesis and its effects on Wnt/β-catenin-VEGF signaling pathway in Lewis lung cancer.Journal of Ethnopharmacology,2016,192:406-412
[7]J-W Zhan,M-S Feng,L-G Zhu,et al.Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in an Organ Culture.BioMed Research International,2016:1-10
[8]M V Risbud,M W Izzo,C S Adams,et al.An organ culture system for the study of the nucleus pulposus:Description of the system and evaluation of the cells.Spine(Phila Pa 1976),2003,28(24):2658-2659
[9]芮泽,张宁,胡志毅,等.持续性压力促进成骨细胞分泌VEGF的机制.南京医科大学学报(自然科学版),2011,7(7):1002-1006
[10]孙旗,柳根哲,贾育松,等.不同静水压对兔椎体终板软骨细胞相关基因表达的影响.中国矫形外科杂志,2014,22(8):753-757
[11]张小海,徐宏光,吴天亮.椎间盘退变与生物力学.国际骨科学杂志,2010,31(3):137-138,161
[12]彭宝淦,贾连顺,施杞,等.软骨终板钙化在椎间盘退变过程中的作用机理.中国矫形外科杂志,2000,7(2):40-43
[13]王弘,陈学武,徐宏光,等.颈椎病终板软骨组织中血管内皮生长因子和缺氧诱导因子-1α表达的相关性.实用医学杂志,2008,24(16):2745-2747
[14]沈颉.血管内皮生长因子(VEGF)在血管外膜成纤维细胞的表达研究.当代医学,2012,18(24):24-26
[15]王弘,陈学武,徐宏光,等.血管内皮生长因子及其受体在颈椎病椎体软骨终板中的表达变化.实用医学杂志,2008,24(8):1336-1338
[16]王拥军,施杞,周重建,等.益气化瘀方对大鼠颈椎间盘软骨终板内血管的影响.中国中医骨伤科杂志,2002,10(4):3-6
[17]Z Li-Guo,F Min-Shan,Z Jia-Wen,et al.Effect of static load on the nucleus pulposus of rabbit intervertebral disc motion segment in ex vivo organ culture.Chinese Medical Journal,2016,129(19):2338-2346
[18]展嘉文,朱立国,冯敏山,等.丹参注射液含药血清对离体培养兔脊柱运动节段椎间盘的影响.中华中医药杂志,2015,30(5):1834-1837
[19]梁广胜,殷明,刘玉亮,等.Wnt信号通路与椎间盘的发展.中国矫形外科杂志,2014,22(5):441-444
[20]任东成,丁金勇.终板软骨细胞退变相关信号通路的研究进展.中国脊柱脊髓杂志,2017,27(7):657-662
[21]陈志刚,吕书军,张建平,等.β连环素在腰椎间盘退变组织中的表达及意义.江苏医药,2015,41(4):408-410
[22]廖军,谢巧瑜,张乐,等.电针对颈椎病模型大鼠椎间盘纤维环细胞Wnt-β-catenin信号通路的影响.中国针灸,2014,34(12):1203-1207
[23]冯敏山,展嘉文,朱立国,等.体外培养条件下兔脊柱运动节段髓核组织的变化.中国组织工程研究,2015,19(51):8241-8246
[24]朱立国,展嘉文,冯敏山,等.兔椎间盘器官与脊柱运动节段离体培养条件下髓核组织的变化.中国骨伤,2015,28(9):824-831
[25]V Easwaran,H L Sang,L Inge,et al.β-Catenin Regulates Vascular Endothelial Growth Factor Expression in Colon Cancer.Cancer Research,2003,63(12):3145
[26]程华,孙芳玲,艾厚喜,等.Wnt信号通路与血管发生研究进展.神经药理学报,2011,1(6):60-63
[27]江华基,江小成,贺飞林,等.黄芩素通过WNT/β-catenin信号通路促进大鼠腱骨愈合.分子影像学杂志,2016,39(3):297-301
[28]余文珍,陈实,张永亮,等.活血化瘀法对鼠颈动脉粥样硬化损伤血管wnt/β-catenin信号通路的影响.世界中医药,2016,11(3):498-501
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