持续压力对离体培养兔椎体终板内Wnt/β-catenin信号通路及VEGF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,每组各8只,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3 kg压力,对照组不予压力,于培养前及培养3、7及14 d时,两组各取10个样本,应用免疫组化、Western blot、Real-time PCR检测VEGF及β-catenin的表达情况。结果免疫组化检测结果显示,持续压力下培养7 d时,终板内VEGF强度显著下降(0.182±0.063),与对照组(0.237±0.051)比较,差异有统计学意义(P<0.05);培养14 d时染色强度进一步降低,压力组(0.156±0.035)与对照组(0.211±0.038)比较,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,培养期间与对照组比较,差异均有统计学意义(P<0.05)。Western blot结果显示,压力组与培养前及对照组比较,上调了β-catenin的表达,差异均有统计学意义(P<0.05)。Real-time PCR结果显示,持续压力导致VEGF基因相对表达量减少(0.842±0.002),与对照组(0.965±0.005)比较,差异有统计学意义(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 study the effect of consistence pressure on the expression of VEGF and beta-catenin in vertebral endplate, and further explore the molecular mechanism between VEGF and intervertebral disc degeneration.Methods A total of 16 New Zealand rabbits were sacrificed after removed under sterile conditions in the spinal motion segment, were randomly divided into control group and pressure group(8 rabbits in each group). Rabbits were placed in vitro loading and culture device for culture, in which the pressure group were treated with consistence pressure 3 kg,the control group did not under pressure, pro-culture and 3, 7, 14 d after culture, 10 samples were collected from each group, the expression of VEGF and beta-catenin were detected by immunohistochemistry(IHC), Western blot and Real time-PCR. Results IHC staining showed that the VEGF staining intensity in the endplate under pressure 7 d after cultured decreased significantly(0.182 ±0.063), compared with the control group(0.237±0.051), the difference was statistically significant(P < 0.05); the staining intensity was further decreased at 14 d after culture(0.156 ±0.035), compared with the control group(0.211±0.038),the difference was statistically significant(P < 0.05).On the contrary, the consistence pressure increased the expression of beta-catenin in the endplate, and compared with that of the control group, the difference was statistically significant(P < 0.05). Western blot results showed that compared with fresh specimen and control group, consistence pressure significantly increased the expression of beta-catenin, the differences were statistically significant(P < 0.05).Real-time PCR results showed the relative expression of VEGF gene was reduced by consistence pressure(0.842 ±0.002), and the difference was significant compared with the control group(0.965±0.005, P < 0.05). Conclusion Con sistence pressure leads to a decrease of VEGF expression and an increase of the expression of beta-catenin in the vertebral endplate of spinal motion segment, which suggests that continuous pressure may regulate the expression of VEGF in the vertebral endplate through the Wnt/beta-catenin signaling pathway.
Objective To study the effect of consistence pressure on the expression of VEGF and beta-catenin in vertebral endplate, and further explore the molecular mechanism between VEGF and intervertebral disc degeneration.Methods A total of 16 New Zealand rabbits were sacrificed after removed under sterile conditions in the spinal motion segment, were randomly divided into control group and pressure group(8 rabbits in each group). Rabbits were placed in vitro loading and culture device for culture, in which the pressure group were treated with consistence pressure 3 kg,the control group did not under pressure, pro-culture and 3, 7, 14 d after culture, 10 samples were collected from each group, the expression of VEGF and beta-catenin were detected by immunohistochemistry(IHC), Western blot and Real time-PCR. Results IHC staining showed that the VEGF staining intensity in the endplate under pressure 7 d after cultured decreased significantly(0.182 ±0.063), compared with the control group(0.237±0.051), the difference was statistically significant(P < 0.05); the staining intensity was further decreased at 14 d after culture(0.156 ±0.035), compared with the control group(0.211±0.038),the difference was statistically significant(P < 0.05).On the contrary, the consistence pressure increased the expression of beta-catenin in the endplate, and compared with that of the control group, the difference was statistically significant(P < 0.05). Western blot results showed that compared with fresh specimen and control group, consistence pressure significantly increased the expression of beta-catenin, the differences were statistically significant(P < 0.05).Real-time PCR results showed the relative expression of VEGF gene was reduced by consistence pressure(0.842 ±0.002), and the difference was significant compared with the control group(0.965±0.005, P < 0.05). Conclusion Con sistence pressure leads to a decrease of VEGF expression and an increase of the expression of beta-catenin in the vertebral endplate of spinal motion segment, which suggests that continuous pressure may regulate the expression of VEGF in the vertebral endplate through the Wnt/beta-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[J].中国中医骨伤科杂志,2016,24(10):70-74.
[2]吕存贤,吴永琴.补阳还五汤对兔退变椎间盘中软骨终板血管芽的影响及意义[J].海峡药学,2012,24(2):33-34.
[3]张巍,徐宏光,俞云飞.短时间机械循环压力对3D培养脊柱终板软骨细胞的影响[J].皖南医学院学报,2015,(1):17-20.
[4]徐坤林,姜宏,刘锦涛.破裂型椎间盘突出动物模型中新生血管因子与炎性反应的研究[J].颈腰痛杂志,2009,30(4):310-312.
[5]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[J].中国骨伤,2017,30(10):926-932.
[6]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[J].中华中医药杂志,2013,(1):108-112.
[7]Han D,Cao C,Su Y,et al.Ginkgo biloba exocarp extracts inhibits angiogenesis and its effects on Wnt/β-catenin-VEGF signaling pathway in Lewis lung cancer[J].J Ethnopharmacol,2016,192:406-412.
[8]Risbud MV,Izzo MW,Adams CS,et al.An organ culture system for the study of the nucleus pulposus:description of the system and evaluation of the cells[J].Spine,2003,28(24):2652-2658.
[9]彭城,任先军,梅芳瑞.椎间盘退变与终板内微血管形态改变的相关性研究[J].中国矫形外科杂志,2003,11(19):1355-1357.
[10]徐鸿明,胡斐,王雍立,等.兔椎体软骨终板内血管三维影像结构研究[J].中国脊柱脊髓杂志,2015,25(11):1013-1017.
[11]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[J].南京医科大学学报:自然科学版,2011,(7):1002-1006.
[12]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[J].中国矫形外科杂志,2014,22(8):753-757.
[13]张小海,徐宏光,吴天亮.椎间盘退变与生物力学[J].国际骨科学杂志,2010,31(3):137-138.
[14]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[J].中国矫形外科杂志,2000,7(2):40-43.
[15]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[J].中国中医骨伤科杂志,2002,10(4):3-6.
[16]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[J].当代医学,2012,18(24):24-26.
[17]Zhu LG,Feng MS,Zhan JW,et al.Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in Ex vivo Organ Culture[J].Chin Med J,2016,129(19):2338-2346.
[18]郭玉生,冯若琳,翁亚菡,等.不同临床分期的子宫内膜癌患者血清VEGF、CRP水平变化情况及相关性研究[J].成都医学院学报,2018,(1):68-70,105.
[19]梁广胜,殷明,刘玉亮,等.Wnt信号通路与椎间盘的发展[J].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[J].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[J].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[J].中国针灸,2014,35(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[J].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[J].中国骨伤,2015,28(9):824-831.
[25]Easwaran V,Lee SH,Inge L,et al.β-Catenin Regulates Vascular Endothelial Growth Factor Expression in Colon Cancer[J].Cancer Res,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[J].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[J].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[J].世界中医药,2016,11(3):498-501.
[2]吕存贤,吴永琴.补阳还五汤对兔退变椎间盘中软骨终板血管芽的影响及意义[J].海峡药学,2012,24(2):33-34.
[3]张巍,徐宏光,俞云飞.短时间机械循环压力对3D培养脊柱终板软骨细胞的影响[J].皖南医学院学报,2015,(1):17-20.
[4]徐坤林,姜宏,刘锦涛.破裂型椎间盘突出动物模型中新生血管因子与炎性反应的研究[J].颈腰痛杂志,2009,30(4):310-312.
[5]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[J].中国骨伤,2017,30(10):926-932.
[6]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[J].中华中医药杂志,2013,(1):108-112.
[7]Han D,Cao C,Su Y,et al.Ginkgo biloba exocarp extracts inhibits angiogenesis and its effects on Wnt/β-catenin-VEGF signaling pathway in Lewis lung cancer[J].J Ethnopharmacol,2016,192:406-412.
[8]Risbud MV,Izzo MW,Adams CS,et al.An organ culture system for the study of the nucleus pulposus:description of the system and evaluation of the cells[J].Spine,2003,28(24):2652-2658.
[9]彭城,任先军,梅芳瑞.椎间盘退变与终板内微血管形态改变的相关性研究[J].中国矫形外科杂志,2003,11(19):1355-1357.
[10]徐鸿明,胡斐,王雍立,等.兔椎体软骨终板内血管三维影像结构研究[J].中国脊柱脊髓杂志,2015,25(11):1013-1017.
[11]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[J].南京医科大学学报:自然科学版,2011,(7):1002-1006.
[12]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[J].中国矫形外科杂志,2014,22(8):753-757.
[13]张小海,徐宏光,吴天亮.椎间盘退变与生物力学[J].国际骨科学杂志,2010,31(3):137-138.
[14]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[J].中国矫形外科杂志,2000,7(2):40-43.
[15]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[J].中国中医骨伤科杂志,2002,10(4):3-6.
[16]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[J].当代医学,2012,18(24):24-26.
[17]Zhu LG,Feng MS,Zhan JW,et al.Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in Ex vivo Organ Culture[J].Chin Med J,2016,129(19):2338-2346.
[18]郭玉生,冯若琳,翁亚菡,等.不同临床分期的子宫内膜癌患者血清VEGF、CRP水平变化情况及相关性研究[J].成都医学院学报,2018,(1):68-70,105.
[19]梁广胜,殷明,刘玉亮,等.Wnt信号通路与椎间盘的发展[J].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[J].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[J].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[J].中国针灸,2014,35(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[J].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[J].中国骨伤,2015,28(9):824-831.
[25]Easwaran V,Lee SH,Inge L,et al.β-Catenin Regulates Vascular Endothelial Growth Factor Expression in Colon Cancer[J].Cancer Res,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[J].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[J].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[J].世界中医药,2016,11(3):498-501.