益气活血方介导促炎因子促进破裂型腰椎间盘突出后重吸收的机制研究
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摘要
目的:通过比较益气活血方介导对趋化因子5(CCL5)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)在破裂及非破裂型髓核细胞中表达的影响,探索益气活血方促进破裂型腰椎间盘突出后重吸收的机制。方法:收集破裂型与非破裂型腰椎间盘突出症患者的髓核组织各6例,采用酶消法分离细胞,放入含20%胎牛血清的DMEM/F12培养基中培养,定期用倒置相差显微镜观察细胞贴壁及生长情况。用10只3个月龄SD大鼠制备含药(益气活血方)血清及无药(生理盐水)血清,用两种血清分别培养破裂型及非破裂髓核细胞。提取两种血清体外给药后的破裂及非破裂原代髓核细胞RNA。利用实时荧光定量PCR(实时qPCR)技术分别检测体外含药血清给药培养后及非含药血清培养后的破裂及非破裂型髓核细胞中CCL5,TNF-α,IL-1βmRNA的表达,记录统计分析数据。提取含药血清及非含药血清体外给药培养后的破裂及非破裂型原代髓核细胞蛋白,利用免疫蛋白印迹(Western Blotting)技术分别检测两种血清体外给药培养后的破裂及非破裂型原代髓核细胞中CCL5,TNF-α及IL-1β蛋白的表达,记录统计分析数据。结果:CCL5,TNF-α及IL-1β在破裂型髓核中的表达明显高于非破裂型(P<0.05),在破裂型含药组髓核细胞中的表达显著高于无药组(P<0.05)。结论:益气活血方可提高促炎因子在破裂型髓核细胞中高表达,有利于髓核细胞的炎性反应,加速细胞外基质(ECM)的降解,从而有利于重吸收现象的发生,且破裂型髓核比非破裂型髓核更容易发生重吸收现象。
Objective:Investigating the mechanism of Yiqi Huoxue recipe in promoting resorption of ruptured lumbar disc herniation,by comparing the expression of CCL5,TNF-α,IL-1β in ruptured and unruptured nucleus pulposus cells mediated by Yiqi Huoxue recipe.Methods:Collecting nucleus pulposus of 6 patients with ruptured lumbar disc herniation and 6 patients with unruptured lumbar disc herniation,separationing of cells by enzymatic elimination,primarying culture in DMEM/F12 medium containing 20%fetal bovine serum,observing cell attachment and growth regular by inverted phase contrast microscope.Ten three-month-old SD rats were prepared for the preparation of drug-containing serum and drugfree serum,cultured ruptured and unruptured nucleus pulposus cells by these serum.Extraction of ruptured and unruptured primary nucleus pulposus cells RNA used two kinds of serum.Detecting the expression of CCL5,TNF-α,IL-1β mRNA in ruptured and unruptured primary nucleus pulposus cells RNA used two kinds of serum by real-time qPCR technology,recording statistical analysis data.Extraction of ruptured and unruptured primary nucleus pulposus cells protein used two kinds of serum,detecting the expression of CCL5,TNF-α,IL-1β mRNA in ruptured and unruptured primary nucleus pulposus cells RNA used two kinds of serum by Western Blotting technology,recording statistical analysis data.Results:The expression of CCL5,TNF-α and IL-1β in ruptured nucleus pulposus was significantly higher than that in unruptured(P<0.05).The expression in the ruptured nucleus pulposus cells of the drug-containing group was significantly higher than that in the drug-free group(P<0.05).Conclusion:Yiqi Huoxue Recipe can increase the expression of pro-inflammatory factors in ruptured nucleus pulposus cells,is useful for inflammatory response of nucleus pulposus cells,can accelerate the degradation of extracellular matrix.Thereby facilitating the occurrence of resorption.The ruptured nucleus pulposus is more prone to resorption than the unruptured nucleus pulposus.
Objective:Investigating the mechanism of Yiqi Huoxue recipe in promoting resorption of ruptured lumbar disc herniation,by comparing the expression of CCL5,TNF-α,IL-1β in ruptured and unruptured nucleus pulposus cells mediated by Yiqi Huoxue recipe.Methods:Collecting nucleus pulposus of 6 patients with ruptured lumbar disc herniation and 6 patients with unruptured lumbar disc herniation,separationing of cells by enzymatic elimination,primarying culture in DMEM/F12 medium containing 20%fetal bovine serum,observing cell attachment and growth regular by inverted phase contrast microscope.Ten three-month-old SD rats were prepared for the preparation of drug-containing serum and drugfree serum,cultured ruptured and unruptured nucleus pulposus cells by these serum.Extraction of ruptured and unruptured primary nucleus pulposus cells RNA used two kinds of serum.Detecting the expression of CCL5,TNF-α,IL-1β mRNA in ruptured and unruptured primary nucleus pulposus cells RNA used two kinds of serum by real-time qPCR technology,recording statistical analysis data.Extraction of ruptured and unruptured primary nucleus pulposus cells protein used two kinds of serum,detecting the expression of CCL5,TNF-α,IL-1β mRNA in ruptured and unruptured primary nucleus pulposus cells RNA used two kinds of serum by Western Blotting technology,recording statistical analysis data.Results:The expression of CCL5,TNF-α and IL-1β in ruptured nucleus pulposus was significantly higher than that in unruptured(P<0.05).The expression in the ruptured nucleus pulposus cells of the drug-containing group was significantly higher than that in the drug-free group(P<0.05).Conclusion:Yiqi Huoxue Recipe can increase the expression of pro-inflammatory factors in ruptured nucleus pulposus cells,is useful for inflammatory response of nucleus pulposus cells,can accelerate the degradation of extracellular matrix.Thereby facilitating the occurrence of resorption.The ruptured nucleus pulposus is more prone to resorption than the unruptured nucleus pulposus.
引文
[1]KADOW T,SOWA G,VO N,et al.Molecular basis of intervertebral disc degeneration and herniations:what are the important translational questions?[J].Clin Orthop Relat Res,2015,473(6):1903-1912.
[2]姜宏,施杞,郑清波.腰椎间盘突出后的自然吸收及其临床意义[J].中华骨科杂志,1998,18(12):755-757.
[3]李晓春,姜宏,刘锦涛.腰椎间盘突出后再吸收的研究进展[J].中国脊柱脊髓杂志,2010,20(7):598-600.
[4]刘锦涛,姜宏,王拥军,等.大鼠破裂型椎间盘突出模型的建立及其重吸收机理的研究[J].中国骨伤,2010,23(5):370-372.
[5]刘锦涛,姜宏,徐坤林,等.非手术疗法对腰椎间盘突出后重吸收的影响[J].中国骨与关节损伤杂志,2010,25(11):978-980.
[6]KANG L,HU J,WENG Y,et al.Sirtuin 6 prevents matrix degradation through inhibition of the NF-κB pathway in intervertebral disc degeneration[J].Experimental Cell Research,2017,352(2):322-332.
[7]ZHOU Z,TIAN FM,WANG P,et al.Alendronate prevents intervertebral disc degeneration adjacent to a lumbar fusion in ovariectomized rats[J].Spine,2015,40(20):1073-1083.
[8]刘锦涛,俞鹏飞,李晓春,等.姜宏教授治疗破裂型腰椎间盘突出症临床经验举隅[J].中国中医骨伤科杂志,2010,18(8):57-58.
[9]LUOMA K,RIIHIMAKI H,LUUKKONEN R,et al.Low back pain in relation to lumbar disc degeneration[J].Spine,2000,25(4):487-492.
[10]MAITRE C L L,BAIRD P,FREEMONT A J,et al.An in vitro study investigating the survival and phenotype of mesenchymal stem cells following injection into nucleus pulposus tissue[J].Arthritis Research&Therapy,2009,11(1):R20.
[11]YASUMA T.Histological changes in aging lumbar intervertebral discs:their role in protrusion and prolapse[J].JBone Joint Surg Am,1990,72(2):220-229.
[12]BINCH A L A,SHAPIRO I M,RISBUD M V.Syndecan-4in intervertebral disc and cartilage:saint or synner?[J].Matrix Biol,2016,s52-54:355-362.
[13]ROBUFFO I,TONIATO E,TETTAMANTI L,et al.Mast cell in innate immunity mediated by proinflammatory and antiinflammatory IL-1family members[J].Journal of Biological Regulators&Homeostatic Agents,2017,31(4):837-842.
[14]OHBA T,HARO H,ANDO T,et al.TNF-alpha-induced NF-kappaB signaling reverses age-related declines in VEGF induction and angiogenic activity in intervertebral disc tissues[J].Journal of Orthopaedic Research,2010,27(2):229-235.
[15]PATTAPPA G,PEROGLIO M,SAKAI D,et al.CCL5/rantes is a key chemoattractant released by degenerative intervertebral discs in organ culture[J].European Cells&Materials,2014,27(1):124-136.
[16]LI P B,TANG W J,WANG K,et al.Expressions of IL-11and MMP-9in degenerated lumbar disc tissues and their clinical significance[J].Eur Rev Med Pharmacol Sci,2017,21(18):4007-4013.
[17]URBAN J P,ROBERTS S.Degeneration of the intervertebral disc[J].Arthritis Research&Therapy,2003,5(3):120-130.
[18]SHAMJI M F,SETTON L A,JARVIS W,et al.Proinflammatory cytokine expression profile in degenerated and herniated human intervertebral disc tissues[J].Arthritis&Rheumatism,2014,62(7):1974-1982.
[19]WEILER C,NERLICH A G,BACHMEIER B E,et al.Expression and distribution of tumor necrosis factor alpha in human lumbar intervertebral discs:a study in surgical specimen and autopsy controls[J].Spine(Phila Pa 1976),2005,30(1):44-53.
[20]LIU X G,HOU H W,LIU Y L.Expression levels of IL-17and TNF-αin degenerated lumbar intervertebral discs and their correlation[J].Experimental&Therapeutic Medicine,2016,11(6):2333-2340.
[21]FUJITA K,ANDO T,OHBA T,et al.Age-related expression of MCP-1and MMP-3in mouse intervertebral disc in relation to TWEAK and TNF-αstimulation[J].Journal of Orthopaedic Research,2012,30(4):599-605.
[22]KIM H J,JIN S Y,KOH Y G,et al.Anti-inflammatory effect of platelet-rich plasma on nucleus pulposus cells with response of TNF-αand IL-1[J].Journal of Orthopaedic Research,2014,32(4):551-556.
[23]HERDER C,ILLIG T,BAUMERT J,et al.RANTES/CCL5gene polymorphisms,serum concentrations,and incident type 2diabetes:results from the MONICA/KORAAugsburg case-cohort study,1984-2002[J].European Journal of Endocrinology,2008,158(5):1-5.
[24]LIOU J T,YUAN H B,MAO C C,et al.Absence of C-Cmotif chemokine ligand 5in mice leads to decreased local macrophage recruitment and behavioral hypersensitivity in a murine neuropathic pain model[J].Pain,2012,153(6):1283-1291.
[25]AHN S H,CHO Y W,AHN M W,et al.mRNA expression of cytokines and chemokines in herniated lumbar intervertebral discs[J].Spine(Phila Pa 1976),2002,27(9):911-917.
[26]KAWAGUCHI S,YAMASHITA T,KATAHIRA G I,et al.Chemokine profile of herniated intervertebral discs infiltrated with monocytes and macrophages[J].Spine(Phila Pa 1976),2002,27(14):1511-1516.
[27]MILLWARDSADLER S J,COSTELLO P W,FREEMONTA J,et al.Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells:implications for the pathogenesis of intervertebral disc degeneration[J].Arthritis Research&Therapy,2009,11(3):R65.
[28]FONG E L S,CHAN C K,GOODMAN S B.Stem cell homing in musculoskeletal injury[J].Biomaterials,2011,32(2):395-409.
[29]KEPLER C K,MARKOVA D Z,DIBRA F,et al.Expression and relationship of proinflammatory chemokine RANTES/CCL5and cytokine IL-1beta in painful human intervertebral discs[J].Spine(Phila Pa 1976),2013,38(11):873-880.
[30]HOYLAND J A,LE MAITRE C,FREEMONT A J.Investigation of the role of IL-1and TNF in matrix degradation in the intervertebral disc[J].Rheumatology,2008,47(6):809-814.
[31]BAEK S J,KANG S K,RA J C.In vitro migration capacity of human adipose tissue-derived mesenchymal stem cells reflects their expression of receptors for chemokines and growth factors[J].Experimental&Molecular Medicine,2011,43(10):596-603.
[2]姜宏,施杞,郑清波.腰椎间盘突出后的自然吸收及其临床意义[J].中华骨科杂志,1998,18(12):755-757.
[3]李晓春,姜宏,刘锦涛.腰椎间盘突出后再吸收的研究进展[J].中国脊柱脊髓杂志,2010,20(7):598-600.
[4]刘锦涛,姜宏,王拥军,等.大鼠破裂型椎间盘突出模型的建立及其重吸收机理的研究[J].中国骨伤,2010,23(5):370-372.
[5]刘锦涛,姜宏,徐坤林,等.非手术疗法对腰椎间盘突出后重吸收的影响[J].中国骨与关节损伤杂志,2010,25(11):978-980.
[6]KANG L,HU J,WENG Y,et al.Sirtuin 6 prevents matrix degradation through inhibition of the NF-κB pathway in intervertebral disc degeneration[J].Experimental Cell Research,2017,352(2):322-332.
[7]ZHOU Z,TIAN FM,WANG P,et al.Alendronate prevents intervertebral disc degeneration adjacent to a lumbar fusion in ovariectomized rats[J].Spine,2015,40(20):1073-1083.
[8]刘锦涛,俞鹏飞,李晓春,等.姜宏教授治疗破裂型腰椎间盘突出症临床经验举隅[J].中国中医骨伤科杂志,2010,18(8):57-58.
[9]LUOMA K,RIIHIMAKI H,LUUKKONEN R,et al.Low back pain in relation to lumbar disc degeneration[J].Spine,2000,25(4):487-492.
[10]MAITRE C L L,BAIRD P,FREEMONT A J,et al.An in vitro study investigating the survival and phenotype of mesenchymal stem cells following injection into nucleus pulposus tissue[J].Arthritis Research&Therapy,2009,11(1):R20.
[11]YASUMA T.Histological changes in aging lumbar intervertebral discs:their role in protrusion and prolapse[J].JBone Joint Surg Am,1990,72(2):220-229.
[12]BINCH A L A,SHAPIRO I M,RISBUD M V.Syndecan-4in intervertebral disc and cartilage:saint or synner?[J].Matrix Biol,2016,s52-54:355-362.
[13]ROBUFFO I,TONIATO E,TETTAMANTI L,et al.Mast cell in innate immunity mediated by proinflammatory and antiinflammatory IL-1family members[J].Journal of Biological Regulators&Homeostatic Agents,2017,31(4):837-842.
[14]OHBA T,HARO H,ANDO T,et al.TNF-alpha-induced NF-kappaB signaling reverses age-related declines in VEGF induction and angiogenic activity in intervertebral disc tissues[J].Journal of Orthopaedic Research,2010,27(2):229-235.
[15]PATTAPPA G,PEROGLIO M,SAKAI D,et al.CCL5/rantes is a key chemoattractant released by degenerative intervertebral discs in organ culture[J].European Cells&Materials,2014,27(1):124-136.
[16]LI P B,TANG W J,WANG K,et al.Expressions of IL-11and MMP-9in degenerated lumbar disc tissues and their clinical significance[J].Eur Rev Med Pharmacol Sci,2017,21(18):4007-4013.
[17]URBAN J P,ROBERTS S.Degeneration of the intervertebral disc[J].Arthritis Research&Therapy,2003,5(3):120-130.
[18]SHAMJI M F,SETTON L A,JARVIS W,et al.Proinflammatory cytokine expression profile in degenerated and herniated human intervertebral disc tissues[J].Arthritis&Rheumatism,2014,62(7):1974-1982.
[19]WEILER C,NERLICH A G,BACHMEIER B E,et al.Expression and distribution of tumor necrosis factor alpha in human lumbar intervertebral discs:a study in surgical specimen and autopsy controls[J].Spine(Phila Pa 1976),2005,30(1):44-53.
[20]LIU X G,HOU H W,LIU Y L.Expression levels of IL-17and TNF-αin degenerated lumbar intervertebral discs and their correlation[J].Experimental&Therapeutic Medicine,2016,11(6):2333-2340.
[21]FUJITA K,ANDO T,OHBA T,et al.Age-related expression of MCP-1and MMP-3in mouse intervertebral disc in relation to TWEAK and TNF-αstimulation[J].Journal of Orthopaedic Research,2012,30(4):599-605.
[22]KIM H J,JIN S Y,KOH Y G,et al.Anti-inflammatory effect of platelet-rich plasma on nucleus pulposus cells with response of TNF-αand IL-1[J].Journal of Orthopaedic Research,2014,32(4):551-556.
[23]HERDER C,ILLIG T,BAUMERT J,et al.RANTES/CCL5gene polymorphisms,serum concentrations,and incident type 2diabetes:results from the MONICA/KORAAugsburg case-cohort study,1984-2002[J].European Journal of Endocrinology,2008,158(5):1-5.
[24]LIOU J T,YUAN H B,MAO C C,et al.Absence of C-Cmotif chemokine ligand 5in mice leads to decreased local macrophage recruitment and behavioral hypersensitivity in a murine neuropathic pain model[J].Pain,2012,153(6):1283-1291.
[25]AHN S H,CHO Y W,AHN M W,et al.mRNA expression of cytokines and chemokines in herniated lumbar intervertebral discs[J].Spine(Phila Pa 1976),2002,27(9):911-917.
[26]KAWAGUCHI S,YAMASHITA T,KATAHIRA G I,et al.Chemokine profile of herniated intervertebral discs infiltrated with monocytes and macrophages[J].Spine(Phila Pa 1976),2002,27(14):1511-1516.
[27]MILLWARDSADLER S J,COSTELLO P W,FREEMONTA J,et al.Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells:implications for the pathogenesis of intervertebral disc degeneration[J].Arthritis Research&Therapy,2009,11(3):R65.
[28]FONG E L S,CHAN C K,GOODMAN S B.Stem cell homing in musculoskeletal injury[J].Biomaterials,2011,32(2):395-409.
[29]KEPLER C K,MARKOVA D Z,DIBRA F,et al.Expression and relationship of proinflammatory chemokine RANTES/CCL5and cytokine IL-1beta in painful human intervertebral discs[J].Spine(Phila Pa 1976),2013,38(11):873-880.
[30]HOYLAND J A,LE MAITRE C,FREEMONT A J.Investigation of the role of IL-1and TNF in matrix degradation in the intervertebral disc[J].Rheumatology,2008,47(6):809-814.
[31]BAEK S J,KANG S K,RA J C.In vitro migration capacity of human adipose tissue-derived mesenchymal stem cells reflects their expression of receptors for chemokines and growth factors[J].Experimental&Molecular Medicine,2011,43(10):596-603.