HIF-1α、MMP-2在退变腰椎间盘髓核中的表达及相关意义
摘要
研究背景
腰椎间盘突出症是脊柱外科最常见的病症之一,导致腰椎间盘突出症的病理基础主要是腰椎间盘退变(intervertebral disc degeneration; IVDD),其表现为凝胶样髓核消失,髓核与纤维环原有清晰界限逐渐消失,纤维环板层粗糙和进行性纤维化。据统计：全世界约80%-90%的人曾经受到过腰腿痛的折磨,其中腰椎间盘退变、突出是最常见的病因,其确切的发病机制尚未完全明确,临床治疗难于达到理想境界。综合既往研究表明,IVDD是多因素协同作用导致的,包括遗传因素、生物力学、生物化学因素等。以基质金属蛋白酶-2(MMP-2)等为代表的基质金属蛋白酶(Matrix metalloproteinase;MMPS)引起基质成分合成、降解紊乱而造成细胞外基质成分的变化是椎间盘力学特征丧失的直接原因之一,进而引起一系列的临床症状。近年来的研究表明缺氧诱导因子-1α(Hypoxia-inducible factor-1α; HIF-1α)在人类突出腰椎间盘中有表达,从而得出结论：HIF-1α在人椎间盘细胞的生成和突出间盘重吸收方面可能起重要作用。在人类的退变椎间盘中HIF-1α与MMP-2的表达有无相关性及它们是如何参与椎间盘的退变过程等方面尚未发现文献报道。有鉴于此,本实验通过对人类正常椎间盘和退变椎间盘组织的对比,应用免疫组织化学染色的方法,观察和分析人类退变椎间盘中的HIF-1α和MMP-2的表达情况。
研究目的
探讨缺氧诱导因子-1α(HIF-1α)、基质金属蛋白酶-2(MMP-2)在人类退变腰椎间盘髓核组织中的表达变化情况；评价HIF-1α、MMP-2在人类腰椎间盘退变过程中的作用,并分析两者之间的关联性。
研究方法
取材均来自南方医科大学第三附属医院脊柱外科2008年1月～2009年6月收集的57例手术标本,分为实验组和对照组：实验组45例,对照组12例。实验组为45例确诊腰椎间盘突出症需手术切除的患者,术中切除的仍位于纤维环内的椎间盘髓核组织,其中男33例,女12例；年龄为20～40岁,平均年龄为31.5岁,病程为3个月～10年。突出部位：L4／L5椎间盘突出23例,L5／S1椎间盘突出20例,L4／L5、L5／S1椎间盘突出2例。根据术前MRI影像表现,对实验组退变腰椎间盘髓核组织分为A、B、C三组,A组：椎间盘信号强度轻度减低,B组：椎间盘信号强度中度减低；C组：椎间盘信号明显减低；对照组12例为正常椎间盘髓核组织,来源于青年脊柱骨折患者行前路减压内固定术时切除的椎间盘髓核组织,术前MRI的T2像见椎间盘呈均匀高信号,其中男10例,女2例；年龄为21-45岁,平均33.7岁。标本取出后均用4%的多聚甲醛固定,而后立即将收集的每个椎间盘髓核组织行石蜡包埋切片,制备5μm的厚组织切片,保存于0～4℃冰箱,HE染色和待免疫组织化学检查。
HIF-1α及MMP-2均购自武汉博士德生物有限公司。具体步骤如下：①石蜡切片常规脱蜡。②3%双氧水室温孵育5-10 min,以消除内源性过氧化物酶的活性。③双氧水冲洗3次,热抗原修复后,PBS(PH7.2-7.6)冲洗3min×2次,滴加5%BSA封闭液,室温20min。甩去多余液体,不洗。④滴加适当稀释一抗(抗人HIF-1α单克隆抗体1：100,或抗人MMP-2单克隆抗体1：100),4℃冰箱过夜。PBS洗2min×3次。⑤滴加生物素化二抗工作液,20～37℃,20分钟。PBS洗2min×3次。⑥滴加试剂SABC,20-37℃,20min。PBS洗5min×3次。⑦显色剂显色(DAB试剂盒)。自来水充分冲洗,复染,封片。显微镜观察。
HIF-1α及MMP-2免疫组化染色都定位于细胞浆,故它们的阳性结果均表现为软骨细胞浆内有棕黄色或棕褐色颗粒。每张切片由两名病理医生在10×20光镜下随机选择10个视野“盲法”阅片,确定细胞总数及阳性表达的细胞数。随机选取10个视野内阳性软骨细胞总数÷软骨细胞总数×100%以计算阳性细胞百分数。采用SPSS 13.0统计软件包对计数资料进行显著性检验和相关性分析。
结果
免疫组化结果为HIF-1α及MMP-2均定位于细胞浆,故显微镜下见HIF-1α及MMP-2的免疫组化阳性结果均表现为软骨细胞胞浆内有棕黄色或棕褐色颗粒,细胞核为淡蓝色。HIF-1α在对照组、轻度退变组、中度退变组、重度退变组椎间盘髓核组织中的表达分别为8.92±3.41%、46.81±3.63%、66.88±4.60%、89.63±2.27%；MMP-2在对照组、轻度退变组、中度退变组、重度退变组椎间盘髓核组织中的表达分别为9.61±3.03%、47.31±4.41%、69.54±5.96%、90.05±3.16%。HIF-1α、MMP-2在实验组退变的椎间盘中的表达显著高于对照组(P<0.05),而且不同退变程度的表达也不同(P<0.05),统计学分析均有显著差异。组间相关系数(r)为0.959,表明HIF-1α和MMP-2在退变的椎间盘组织中的表达呈正相关的关系。
结论
HIF-1α、MMP-2均可能参与了人类腰椎间盘组织的退变过程,且HIF-1α、MMP-2在人类退变腰椎间盘组织中有相同的增高趋势。HIF-1α可能与MMP-2相互协调、共同促进椎间盘退变。这些方面的深入研究及有益尝试将为预防和治疗腰椎间盘髓核退变提供新的思路。
Background data
The lumber disc hernia(LDH) is the leading disease in the spine surgery. Intervertebral disc degeneration(IVDD) is the main pathological reason. the dehydration of the gel-like nucleus pulposus, progressive fibrosis of the annulus fibrosus has been presented in the aging.Nucleus pulposus and annulus fibrosus gradually disappearing boundaries of the original definition. In fact, almost 80% of adults will encounter some form of back pain in their lifetimes. The degeneration of the disc is the leading cause of back pain. But its exact pathogenesis has not yet clearly.Past studies have shown that Genetic factors,biomechanics and biochemistry elements are relevant with IVDD。
Recently, the fundamental research of disc degeneration has been focused on immunology and genetic aspects,the immunological research of Matrix metalloproteinase investigated that the disorder of synthesis and degradation of the matrix caused by MMPS is one of the reasons for the loss of the disc biomechanics, the expression of Hypoxia-inducible factor-1α(HIF-1α) have been detected in the herniated disc of the human beings in current report. HIF-1αcould be another impact factor on the reabsorption and the generation of the disc cells. No paper about the correlation between HIF-1αand MMP-2, the mechanism of the two factor involved in the process of disc degeneration, has been published. We performed the comparison of the expression of the HIF-la and MMP-2 in both normal and degenerated disc to reveal the disc degeneration further.
Objective
To investigate the expression of the HIF-la and MMP-2 in the disc with different degree of degeneration and to evaluate the significance and the correlation of the two factors in the process of the degeneration。
Materials and Methods
All the materials were drawn from the spinal surgery departments of the third affiliated hospitaof the Southern Medical University during jan,2008 to june,2009.Experimental group contained 45 cases diagnosed as LDH,Male 33 cases, female 12cases. The mean age is 31.5 years old(range from 20 to 40), the course of the disease range form 3 months to 10years;L4/L5 disc herniation in 23 cases, L5/S1 disc herniation 20 cases, L4/L5, L5/S1 disc herniation in 2 cases. Nucleus pulposus inside the annulus fibrosis were removed as main materials. Based on preoperative MRI imaging, the degenerated nucleus pulposus has been divided into 3 groups. Group A:Disc signal intensity slightly reduced, Group B:Disc signal intensity moderate reduced; Group C: Disc signal intensity disappeared. Control group included 12cases,male 10 cases, female 2 cases, The mean age is 31.5 years old(range from 20 to 40).The normal nucleus pulposus which was removed during the anterior decompression was obtained from young patients with spinal fracture. T2 weighted imaging presented disc uniform high signal intensity on preoperative MRI imaging.
All samples were rinsed thoroughly in 0.9%NaCl to remove blood right after the surgical Procedure, and soaped in 4% formalin within 3 min.After fixed for 24 hours, samples were embedded in paraffin wax, then sectioned to 5 um thick slices.Immunostaining method was used to detect the expression level of HIF-1αand MMP2.Since HIF-1αand MMP2 are located in the cytoplasm,the presence of these proteins is correlated with brown grains in the cytosol when observed under microseope. The cell counting was performed under the microscope by two Pathologist.10 fields were randomly selected and the number of cells was counted.protein expression level was showed as the percentage of positive staining cells. All the data were analyzed by SPSS 13.0 software package for the significance test and the correlation analysis. The P value was set at 0.05.
Result
The result of the immunohistochemical test showed HIF-1αand MMP-2 were sited at the cytoplasm, the positive result of the cartilage cell displayed the Brown yellow or brown granules in the cytoplasm, the coloring of the nucleolus is light blue. The expression of HIF-1α、MMP-2 in the experimental group is significant higher than the control group (P<0.05),meanwhile, the expression in the disc with different degree of degeneration varied (P<0.05) it is significant difference.r=0.959, the expression of the HIF-1αand MMP-2 in the degeneration of the intervertebral disc was positively correlated.
Conclusions
HIF-1αand MMP-2 are both important impact factors involved in the degeneration of the human beings lumber disc. The expression of the HIF-1αand MMP-2 in the degeneration of the disc was positively correlated. With the coordinating of MMP-2 HIF-1αcould be a regulated element of MMP-2 to promote the intervertebral disc degeneration.
腰椎间盘突出症是脊柱外科最常见的病症之一,导致腰椎间盘突出症的病理基础主要是腰椎间盘退变(intervertebral disc degeneration; IVDD),其表现为凝胶样髓核消失,髓核与纤维环原有清晰界限逐渐消失,纤维环板层粗糙和进行性纤维化。据统计：全世界约80%-90%的人曾经受到过腰腿痛的折磨,其中腰椎间盘退变、突出是最常见的病因,其确切的发病机制尚未完全明确,临床治疗难于达到理想境界。综合既往研究表明,IVDD是多因素协同作用导致的,包括遗传因素、生物力学、生物化学因素等。以基质金属蛋白酶-2(MMP-2)等为代表的基质金属蛋白酶(Matrix metalloproteinase;MMPS)引起基质成分合成、降解紊乱而造成细胞外基质成分的变化是椎间盘力学特征丧失的直接原因之一,进而引起一系列的临床症状。近年来的研究表明缺氧诱导因子-1α(Hypoxia-inducible factor-1α; HIF-1α)在人类突出腰椎间盘中有表达,从而得出结论：HIF-1α在人椎间盘细胞的生成和突出间盘重吸收方面可能起重要作用。在人类的退变椎间盘中HIF-1α与MMP-2的表达有无相关性及它们是如何参与椎间盘的退变过程等方面尚未发现文献报道。有鉴于此,本实验通过对人类正常椎间盘和退变椎间盘组织的对比,应用免疫组织化学染色的方法,观察和分析人类退变椎间盘中的HIF-1α和MMP-2的表达情况。
研究目的
探讨缺氧诱导因子-1α(HIF-1α)、基质金属蛋白酶-2(MMP-2)在人类退变腰椎间盘髓核组织中的表达变化情况；评价HIF-1α、MMP-2在人类腰椎间盘退变过程中的作用,并分析两者之间的关联性。
研究方法
取材均来自南方医科大学第三附属医院脊柱外科2008年1月～2009年6月收集的57例手术标本,分为实验组和对照组：实验组45例,对照组12例。实验组为45例确诊腰椎间盘突出症需手术切除的患者,术中切除的仍位于纤维环内的椎间盘髓核组织,其中男33例,女12例；年龄为20～40岁,平均年龄为31.5岁,病程为3个月～10年。突出部位：L4／L5椎间盘突出23例,L5／S1椎间盘突出20例,L4／L5、L5／S1椎间盘突出2例。根据术前MRI影像表现,对实验组退变腰椎间盘髓核组织分为A、B、C三组,A组：椎间盘信号强度轻度减低,B组：椎间盘信号强度中度减低；C组：椎间盘信号明显减低；对照组12例为正常椎间盘髓核组织,来源于青年脊柱骨折患者行前路减压内固定术时切除的椎间盘髓核组织,术前MRI的T2像见椎间盘呈均匀高信号,其中男10例,女2例；年龄为21-45岁,平均33.7岁。标本取出后均用4%的多聚甲醛固定,而后立即将收集的每个椎间盘髓核组织行石蜡包埋切片,制备5μm的厚组织切片,保存于0～4℃冰箱,HE染色和待免疫组织化学检查。
HIF-1α及MMP-2均购自武汉博士德生物有限公司。具体步骤如下：①石蜡切片常规脱蜡。②3%双氧水室温孵育5-10 min,以消除内源性过氧化物酶的活性。③双氧水冲洗3次,热抗原修复后,PBS(PH7.2-7.6)冲洗3min×2次,滴加5%BSA封闭液,室温20min。甩去多余液体,不洗。④滴加适当稀释一抗(抗人HIF-1α单克隆抗体1：100,或抗人MMP-2单克隆抗体1：100),4℃冰箱过夜。PBS洗2min×3次。⑤滴加生物素化二抗工作液,20～37℃,20分钟。PBS洗2min×3次。⑥滴加试剂SABC,20-37℃,20min。PBS洗5min×3次。⑦显色剂显色(DAB试剂盒)。自来水充分冲洗,复染,封片。显微镜观察。
HIF-1α及MMP-2免疫组化染色都定位于细胞浆,故它们的阳性结果均表现为软骨细胞浆内有棕黄色或棕褐色颗粒。每张切片由两名病理医生在10×20光镜下随机选择10个视野“盲法”阅片,确定细胞总数及阳性表达的细胞数。随机选取10个视野内阳性软骨细胞总数÷软骨细胞总数×100%以计算阳性细胞百分数。采用SPSS 13.0统计软件包对计数资料进行显著性检验和相关性分析。
结果
免疫组化结果为HIF-1α及MMP-2均定位于细胞浆,故显微镜下见HIF-1α及MMP-2的免疫组化阳性结果均表现为软骨细胞胞浆内有棕黄色或棕褐色颗粒,细胞核为淡蓝色。HIF-1α在对照组、轻度退变组、中度退变组、重度退变组椎间盘髓核组织中的表达分别为8.92±3.41%、46.81±3.63%、66.88±4.60%、89.63±2.27%；MMP-2在对照组、轻度退变组、中度退变组、重度退变组椎间盘髓核组织中的表达分别为9.61±3.03%、47.31±4.41%、69.54±5.96%、90.05±3.16%。HIF-1α、MMP-2在实验组退变的椎间盘中的表达显著高于对照组(P<0.05),而且不同退变程度的表达也不同(P<0.05),统计学分析均有显著差异。组间相关系数(r)为0.959,表明HIF-1α和MMP-2在退变的椎间盘组织中的表达呈正相关的关系。
结论
HIF-1α、MMP-2均可能参与了人类腰椎间盘组织的退变过程,且HIF-1α、MMP-2在人类退变腰椎间盘组织中有相同的增高趋势。HIF-1α可能与MMP-2相互协调、共同促进椎间盘退变。这些方面的深入研究及有益尝试将为预防和治疗腰椎间盘髓核退变提供新的思路。
Background data
The lumber disc hernia(LDH) is the leading disease in the spine surgery. Intervertebral disc degeneration(IVDD) is the main pathological reason. the dehydration of the gel-like nucleus pulposus, progressive fibrosis of the annulus fibrosus has been presented in the aging.Nucleus pulposus and annulus fibrosus gradually disappearing boundaries of the original definition. In fact, almost 80% of adults will encounter some form of back pain in their lifetimes. The degeneration of the disc is the leading cause of back pain. But its exact pathogenesis has not yet clearly.Past studies have shown that Genetic factors,biomechanics and biochemistry elements are relevant with IVDD。
Recently, the fundamental research of disc degeneration has been focused on immunology and genetic aspects,the immunological research of Matrix metalloproteinase investigated that the disorder of synthesis and degradation of the matrix caused by MMPS is one of the reasons for the loss of the disc biomechanics, the expression of Hypoxia-inducible factor-1α(HIF-1α) have been detected in the herniated disc of the human beings in current report. HIF-1αcould be another impact factor on the reabsorption and the generation of the disc cells. No paper about the correlation between HIF-1αand MMP-2, the mechanism of the two factor involved in the process of disc degeneration, has been published. We performed the comparison of the expression of the HIF-la and MMP-2 in both normal and degenerated disc to reveal the disc degeneration further.
Objective
To investigate the expression of the HIF-la and MMP-2 in the disc with different degree of degeneration and to evaluate the significance and the correlation of the two factors in the process of the degeneration。
Materials and Methods
All the materials were drawn from the spinal surgery departments of the third affiliated hospitaof the Southern Medical University during jan,2008 to june,2009.Experimental group contained 45 cases diagnosed as LDH,Male 33 cases, female 12cases. The mean age is 31.5 years old(range from 20 to 40), the course of the disease range form 3 months to 10years;L4/L5 disc herniation in 23 cases, L5/S1 disc herniation 20 cases, L4/L5, L5/S1 disc herniation in 2 cases. Nucleus pulposus inside the annulus fibrosis were removed as main materials. Based on preoperative MRI imaging, the degenerated nucleus pulposus has been divided into 3 groups. Group A:Disc signal intensity slightly reduced, Group B:Disc signal intensity moderate reduced; Group C: Disc signal intensity disappeared. Control group included 12cases,male 10 cases, female 2 cases, The mean age is 31.5 years old(range from 20 to 40).The normal nucleus pulposus which was removed during the anterior decompression was obtained from young patients with spinal fracture. T2 weighted imaging presented disc uniform high signal intensity on preoperative MRI imaging.
All samples were rinsed thoroughly in 0.9%NaCl to remove blood right after the surgical Procedure, and soaped in 4% formalin within 3 min.After fixed for 24 hours, samples were embedded in paraffin wax, then sectioned to 5 um thick slices.Immunostaining method was used to detect the expression level of HIF-1αand MMP2.Since HIF-1αand MMP2 are located in the cytoplasm,the presence of these proteins is correlated with brown grains in the cytosol when observed under microseope. The cell counting was performed under the microscope by two Pathologist.10 fields were randomly selected and the number of cells was counted.protein expression level was showed as the percentage of positive staining cells. All the data were analyzed by SPSS 13.0 software package for the significance test and the correlation analysis. The P value was set at 0.05.
Result
The result of the immunohistochemical test showed HIF-1αand MMP-2 were sited at the cytoplasm, the positive result of the cartilage cell displayed the Brown yellow or brown granules in the cytoplasm, the coloring of the nucleolus is light blue. The expression of HIF-1α、MMP-2 in the experimental group is significant higher than the control group (P<0.05),meanwhile, the expression in the disc with different degree of degeneration varied (P<0.05) it is significant difference.r=0.959, the expression of the HIF-1αand MMP-2 in the degeneration of the intervertebral disc was positively correlated.
Conclusions
HIF-1αand MMP-2 are both important impact factors involved in the degeneration of the human beings lumber disc. The expression of the HIF-1αand MMP-2 in the degeneration of the disc was positively correlated. With the coordinating of MMP-2 HIF-1αcould be a regulated element of MMP-2 to promote the intervertebral disc degeneration.
引文
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[9]Cooke PM, Lutz GE. Internal disc disruption and axial back pain in the athlete. Phys Med Rehabil Clin N Am.2000,11:837-65.
[10]Elfering A, Semmer N, Birkhofer D,et al. Risk factors for lumbar disc degeneration: a 5-year prospective MRI study in asymptomatic individuals. Spine.2002; 27:125-34.
[11]Seidler A, Bolm-Audorff U, Siol T, et al.Occupational risk factors for symptomatic lumbar disc herniation; a case-control study. Occupational and environmental medicine 2003,60:821-830.
[12]Spiliopoulou I,Korovessis P,Konstantinou D,et al.IgG and IgMconcentration in the prolapsed human intervertebral disc andsciatica etiology[J] Spine,1994,19(12): 1320-1323.
[13]Kang JD, Georgescu HI, McIntyre-Larkin L, et al.Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 [J].Spine,1996,21:271-277.
[14]Takahashi H,Suguro T,Okazima Y,et al.Inflammatory cytokinesin the herniated disc of the lumbar spine[J].Spine,1996,21:218-224.
[15]Sedowofia KA, Tonlision IW, Weiss JB, et al.Collagenolytic enzyme systems in human intervertebral disc:theireontrol, eehanism, and their Possible role in the inition of biochemieal failure SPine,1982,7:213-222.
[16]HaKY, Koh IJ, Kirpalani PA, et al·The expression of hypoxia inducible factor-lalpha and apoptosis in herniated discs-Spine,2006,31 (12):1309-1313.
[17]龙厚清,李佛保,刘少喻,等.腰椎间盘退变的分期及其病理学[J].脊柱外科杂志,2004,2(4)：234-236.
[18]Videman T,Nummi P,Battie NC,et al. Digital assessment of MRI for lumbar disc desiccation:a comparison of digital versus subjective assessment and digital intensity profiles versus discogram and macroanatomic findings [J].spine.1994, 19;192-198.
[19]Niemelainen R,Videman T,Dhillon SS,et al. Quantitative measurement of intervertebral disc signal using MRI. Clinical Radiology 2008;63(3):252-5.
[20]Thelander J,Fagerlund M,Fribergs,et al. Straight leg raising test VS.radiologic size,shape,and position of lumbar disc herniations [J].SPine,1992,17:395-399.
[21]Takahashi H,Suguro T,Okazima Y,et al.Inflammatory cytokinesin the herniated disc of the lumbar spine[J].Spine,1996,21:218-224.
[22]Burke JQWatson RW,McCormack D,et al.Intervertebral discs which cause low back pain secrete high levels of proinflammatory mediators [J].J Bone Joint Surg Br,2002,84 (2):196-201.
[23]Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol.1992,12:5447-5454.
[24]Zheng JZ, Leung SW, Weisz A, et al.Transactivation and inhibitory domains of hypoxia-inducible factor 1 alpha modulation of transcriptional activity by oxygen tension.J BiolChem,1997,272 (31):19253-19260.
[25]Pugh CW,Ratclife PJ.Regulation of angiogenesis by hypoxia:role of the HIF system.NatMed,2003,9:677-684.
[26]Hamacher S, Matern S, Roeb E.Extracellular matrix-from basic research to clinical significance:an overview with special consideration of matrix metalloproteinases J.Dtsch Med Wochenschr.2004,129-(38):1976-1980.
[27]Goupille P,Jayson M I, ValatJ P.et al.Matrix Metalloproteinases:The Clue to Intervertebral Disc Degeneration?[J]SPine,1998,15,23(14):1612-26.
[28]Nerlich AG,Schleicher ED,Boos N.Immunohistologic markers for age-relatned changes ofhuman lumbar intervertebral discs.Spine,1997,22:2781-95.
[29]Roberts S,Caterson B,Menage J,etal. Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc. [J]. Spine,2000, 25(23):3005-3013.
[30]Krishnamachary B,Berg-Dixons, Kelly B,et al.Regulation of colon carcinoma cell invasion by hypoxia-inducible factor-1 [J].Cancer Res,2003,63(5):1138-1143.
[1]Naylar A. Enzymic and immunological activity in the intervertebral disc.Orthop Clin N Am,1975,6,51.
[2]Spiliopoulou I,Korovessis P,Konstantinou D,et al.IgG and IgMconcentration in the prolapsed human intervertebral disc andsciatica etiology[J]. Spine,1994,19(12): 1320-1323.
[3]马信龙,徐云强,张义修,等.腰椎间盘突出症自身免疫因素的研究[J].中国现代神经疾病杂志,2004,4(5)：291—296.
[4]王葵光,胡有谷.腰椎间盘突出症的自身免疫状态.中华骨科杂志,1994,14：258～262.
[5]Kang JD, Georgescu HI, McIntyre-Larkin L, et al.Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 [J].Spine,1996,21:271-277.
[6]Takahashi H,Suguro T,Okazima Y,et al.Inflammatory cytokinesin the herniated disc of the lumbar spine[J].Spine,1996,21:218-224.
[7]Sedowofia KA, Tonlision IW, Weiss JB, et al.Collagenolytic enzyme systems in human intervertebral disc:theireontrol, eehanism, and their Possible role in the inition of biochemieal failure SPine,1982,7:213-222.
[8]HaKY, Koh IJ, Kirpalani PA, et al-The expression of hypoxia inducible factor-lalpha and apoptosis in herniated discs-Spine,2006,31 (12):1309-1313.
[9]Burke JG, Watson RW,McCormack D,et al.Intervertebral discs which cause low back pain secrete high levels of proinflammatory mediators[J].J Bone Joint Surg Br,2002,84 (2):196-201.
[10]Mizel SB, dayer JM, Krane SM, et al. Stimmulation of rheumatoid synovial cellcollagenase and prostaylandin production by partially purified lymphocyte activitiny faotor (interleukin-l), procNatIAeal SciUSA,1981,17—8:2474～2477.
[11]O'Donnell JL,O'Donnell AL.Prostaglandin E2 content in herniated lumbar disc diease. Spine,1996,21:1653～1656.
[12]赵剑,赵敦炎,张明,等.白细胞介素-1在突出腰椎间盘中的表达[J].中国脊柱脊髓杂志,2000,10：15-17.
[13]Rannou F,Corvol MT,Hudry C,et al.Sensitivity of anulus fibrosus cells to interleukin 1 beta.Comparison with articularchondrocytes[J].Spine,2000,25(1): 17—23.
[14]龚非力.医学免疫学第二版.北京：科学出版社,2004,6.
[15]Pelletier JP, Dibattishta JA, Rouyhley P,et al. R beum Dis, Clin North(Am),1993, 19(3):545～568.
[16][Kang JD,Stefanovic(Racic M,McIntyre LA,et al.Toward a biochemical understanding of human intervertebral disc degeneration and herniation: Contributions of nitric oxide, interleukins,prostaglandin E2,and matrix metalloproteinases[J].Spine,1997,22:1065-1073.
[17]李新友,王民,刘淼,等.白细胞介素-6在突出的腰椎间盘中的表达及其意义.中国脊柱脊髓杂志,2001,8：581-582.
[18]Burke JG, G Watson RW, Conhyea D, et al. Human Nucleus Pulposis Can Respond to a Pro-inflammatory Stimulus[J]. Spine,2003,28(24):2685-269.
[19][赵太茂,刘森,宋红星,等.突出椎间盘组织中TNF-a与IL-b的表达及意义[J].中国脊柱脊髓杂志,1999,9(1)：17.
[20]Valdes,AnaM,Hassett, Geraldine MBBS. Radiographic Progression of Lumbar Spine Disc Degeneration Is Influenced by Variation at Inflammatory Genes:A Candidate SNPAssociation Study in the Chingford Cohort[J]. spine,2005,30(21): 2445.
[21]Rand N,Reichert F,Floman Y,et al.Murine nucleus pulposus-derived cells secrete interleukins-1-beta,-6,and-10 and granulocyte-macrophage colony-stimulating factor in cell culture.Spine 1997;22(22):2598-2601.
[22]Kato T,Haro H,Komori H,et al.Sequential dynamics of inflammatory cytokine,angiogenesis inducing factor and matrix degrading enzymes during spontaneous resorption of the herniated disc[J].J Orthop Res,2004,22(4):895—900.
[23]YamanakaH, MakinoK, TakizawaM, et al.Expression and tissue localization of membrane-types 1,2, and 3 matrixmetalloproteinases in rheumatoid synovium. Lab Invest,2000,80:677-687.
[24]Hammacher S,Matern S,Rroeb E.Extracellular matrix-form basic research to clinical significance:an overview with special consideration of metalloproteinases J.Dtsch Med Wochenschr-.2004,129-(38):1976-1980.
[25]Massova I, Kotra LP, Fridman R, eta.l. Matrixmetalloproteinases:structures, evolution, and diversification. FASEB J,1998,12:1075-1095.
[26]Gardner J,Ghorpade A.Tissue inhibitor of metalloproteinase (TIMP)-1:the TIMPed balance of matrix metalloproteinases in the central nervous system[J].J Neurosci Res,2003,74(6):801-806.
[27]Weiler C,Nerlich AG,Zipperer J,et al.2002 SSE Award Competition in Basic Science:expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorption[J].Eur Spine J,2002,11(4):308-320.
[28]Roberts S,Caterson B,Menage J,et al.Matrix metalloproteinases and aggrecanase:their role in disorders of the human intervertebral disc [J]. Spine, 2000,25(23):3005-3013.
[29]Kanemoto M,Hukuda S,Komiya Y,et al.Immunohistochemical study of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 human intervertebral discs[J].Spine,1996,21(1):1-8.
[30]Semenza GL, Wang GL·A nuclear factor induced by hy-poxia via denovo protein synthesis binds to the human erythropoietin gene enhancer at a site required for tran-scriptional activation-Mol Cell Bio,l 1992,12:5447-5454.
[31]Zheng JZ, Leung SW, Weisz A, et al·Transactivation and inhibitory domains of hypoxia-inducible factor lalpha modulation of transcriptional activity by oxygen tension·J BiolChem,1997,272 (31):19253-19260.
[32]Pugh CW, Ratclife PJ·Regulation of angiogenesis by hy-poxia:role of theHIF system-NatMed,2003,9:677-68.
[33]王俊丰,贾长青,刘洪亮等.缺氧诱导因子-1α和血管内皮生长因子在突出腰椎间盘中的表达及意义[J].中国组织化学与细胞化学杂志,2008,17(1)：65-69.
[34]Liu GZ,Ishihara H,Osada R,et al.Nitric oxide mediates the change of proteoglycan synthesis in the human lumbar intervertebral disc in response to hydrostatic pressure[J]. Spine,2001,26:134-141.
[35]Kohyama K,Saura R,Doita M, et al. Intervertebral disc cell apoptosis by nitric oxide:Biological understanding of intervertebral disc degeneration[J].Kobe J Med Sci,2000,46:283-295.
[36]Furusawa N,Baba H,Miyoshi N,et al.Herniation of cervical intervertebral disc:immunohistochemical examination and measurement of nitric oxide production[J].Spine,2001,26(10):1110—1116.
[37]李小川,李雷,王欢.Fas/FasL基因在退变腰椎间盘组织中的表达及诱导凋亡作用[J].中华医学杂志,2005,85(24)：1718-1720.
[38]吕国华,李晶,周江南,等.腰椎间盘组织的炎症诱导效应的实验研究[J].湖南医科大学学报,2001,26(6)：532.
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[41]叶君健,宋继红.腰椎间盘突出症髓核组织磷脂酶A2表达的临床研究[J].中国骨与关节损伤杂志,2006,21(4)：259-261.
[42]Nishida T.Kinetics of tissue and serum matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 in intervertebral disc degeneration and disc herniation.Kurume Med J.1999;46(1):39-50.
[43]郑煜新,石印玉.腰椎间盘突出症血清MMP-3测定的意义.颈腰痛杂志,2001,22：19～20.
[44]于杰,朱立国,高景华,等.腰椎间盘突出症患者血清白细胞介素1β、白细胞介素6、肿瘤坏死因子α表达与其疼痛的相关性[J].中国组织工程研究与临床康复,2007,11(2)：301-304.
[2]Diwan AD,Parvataneni HK,Khan SN,et al.Current concepts in intervertebral disc restoration.Orthop Clin North Am,2000,31:453-464.
[3]Beatty RA.Myxomatous degeneration of the lumbar intervertebral disc.Neurosurgery,1985,17:277-280.
[4]Natarajan RN,Ke JH,Andersson GB.A model to study the disc degeneration process.Spine,1994,19:259-265.
[5]Weiler C, Nerlich AG, Zipperer J, et al.2002 SSE award competition in basic science expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorptionp[J].Eur Spine J,2002, 11(4):308-320.
[6]JK Richardson, T Chung, JS Schultz,et al. A familial predisposition toward lumbar disc injury.Spine,1997:22:1487-1492.
[7]MC Battie,DR Haynor,LD Fisher.et al.Similarities in degenerative findings on magnetic resonance images of the lumbar spines of identical twins. The Journal of Bone and Joint Surgery,1995:77,1662-1670.
[8]Saicheua P. Occupational lumbar disc herniation among Thai workers claimed for compensation. J Med Assoc Thai.2001:84:253-7.
[9]Cooke PM, Lutz GE. Internal disc disruption and axial back pain in the athlete. Phys Med Rehabil Clin N Am.2000,11:837-65.
[10]Elfering A, Semmer N, Birkhofer D,et al. Risk factors for lumbar disc degeneration: a 5-year prospective MRI study in asymptomatic individuals. Spine.2002; 27:125-34.
[11]Seidler A, Bolm-Audorff U, Siol T, et al.Occupational risk factors for symptomatic lumbar disc herniation; a case-control study. Occupational and environmental medicine 2003,60:821-830.
[12]Spiliopoulou I,Korovessis P,Konstantinou D,et al.IgG and IgMconcentration in the prolapsed human intervertebral disc andsciatica etiology[J] Spine,1994,19(12): 1320-1323.
[13]Kang JD, Georgescu HI, McIntyre-Larkin L, et al.Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 [J].Spine,1996,21:271-277.
[14]Takahashi H,Suguro T,Okazima Y,et al.Inflammatory cytokinesin the herniated disc of the lumbar spine[J].Spine,1996,21:218-224.
[15]Sedowofia KA, Tonlision IW, Weiss JB, et al.Collagenolytic enzyme systems in human intervertebral disc:theireontrol, eehanism, and their Possible role in the inition of biochemieal failure SPine,1982,7:213-222.
[16]HaKY, Koh IJ, Kirpalani PA, et al·The expression of hypoxia inducible factor-lalpha and apoptosis in herniated discs-Spine,2006,31 (12):1309-1313.
[17]龙厚清,李佛保,刘少喻,等.腰椎间盘退变的分期及其病理学[J].脊柱外科杂志,2004,2(4)：234-236.
[18]Videman T,Nummi P,Battie NC,et al. Digital assessment of MRI for lumbar disc desiccation:a comparison of digital versus subjective assessment and digital intensity profiles versus discogram and macroanatomic findings [J].spine.1994, 19;192-198.
[19]Niemelainen R,Videman T,Dhillon SS,et al. Quantitative measurement of intervertebral disc signal using MRI. Clinical Radiology 2008;63(3):252-5.
[20]Thelander J,Fagerlund M,Fribergs,et al. Straight leg raising test VS.radiologic size,shape,and position of lumbar disc herniations [J].SPine,1992,17:395-399.
[21]Takahashi H,Suguro T,Okazima Y,et al.Inflammatory cytokinesin the herniated disc of the lumbar spine[J].Spine,1996,21:218-224.
[22]Burke JQWatson RW,McCormack D,et al.Intervertebral discs which cause low back pain secrete high levels of proinflammatory mediators [J].J Bone Joint Surg Br,2002,84 (2):196-201.
[23]Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol.1992,12:5447-5454.
[24]Zheng JZ, Leung SW, Weisz A, et al.Transactivation and inhibitory domains of hypoxia-inducible factor 1 alpha modulation of transcriptional activity by oxygen tension.J BiolChem,1997,272 (31):19253-19260.
[25]Pugh CW,Ratclife PJ.Regulation of angiogenesis by hypoxia:role of the HIF system.NatMed,2003,9:677-684.
[26]Hamacher S, Matern S, Roeb E.Extracellular matrix-from basic research to clinical significance:an overview with special consideration of matrix metalloproteinases J.Dtsch Med Wochenschr.2004,129-(38):1976-1980.
[27]Goupille P,Jayson M I, ValatJ P.et al.Matrix Metalloproteinases:The Clue to Intervertebral Disc Degeneration?[J]SPine,1998,15,23(14):1612-26.
[28]Nerlich AG,Schleicher ED,Boos N.Immunohistologic markers for age-relatned changes ofhuman lumbar intervertebral discs.Spine,1997,22:2781-95.
[29]Roberts S,Caterson B,Menage J,etal. Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc. [J]. Spine,2000, 25(23):3005-3013.
[30]Krishnamachary B,Berg-Dixons, Kelly B,et al.Regulation of colon carcinoma cell invasion by hypoxia-inducible factor-1 [J].Cancer Res,2003,63(5):1138-1143.
[1]Naylar A. Enzymic and immunological activity in the intervertebral disc.Orthop Clin N Am,1975,6,51.
[2]Spiliopoulou I,Korovessis P,Konstantinou D,et al.IgG and IgMconcentration in the prolapsed human intervertebral disc andsciatica etiology[J]. Spine,1994,19(12): 1320-1323.
[3]马信龙,徐云强,张义修,等.腰椎间盘突出症自身免疫因素的研究[J].中国现代神经疾病杂志,2004,4(5)：291—296.
[4]王葵光,胡有谷.腰椎间盘突出症的自身免疫状态.中华骨科杂志,1994,14：258～262.
[5]Kang JD, Georgescu HI, McIntyre-Larkin L, et al.Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2 [J].Spine,1996,21:271-277.
[6]Takahashi H,Suguro T,Okazima Y,et al.Inflammatory cytokinesin the herniated disc of the lumbar spine[J].Spine,1996,21:218-224.
[7]Sedowofia KA, Tonlision IW, Weiss JB, et al.Collagenolytic enzyme systems in human intervertebral disc:theireontrol, eehanism, and their Possible role in the inition of biochemieal failure SPine,1982,7:213-222.
[8]HaKY, Koh IJ, Kirpalani PA, et al-The expression of hypoxia inducible factor-lalpha and apoptosis in herniated discs-Spine,2006,31 (12):1309-1313.
[9]Burke JG, Watson RW,McCormack D,et al.Intervertebral discs which cause low back pain secrete high levels of proinflammatory mediators[J].J Bone Joint Surg Br,2002,84 (2):196-201.
[10]Mizel SB, dayer JM, Krane SM, et al. Stimmulation of rheumatoid synovial cellcollagenase and prostaylandin production by partially purified lymphocyte activitiny faotor (interleukin-l), procNatIAeal SciUSA,1981,17—8:2474～2477.
[11]O'Donnell JL,O'Donnell AL.Prostaglandin E2 content in herniated lumbar disc diease. Spine,1996,21:1653～1656.
[12]赵剑,赵敦炎,张明,等.白细胞介素-1在突出腰椎间盘中的表达[J].中国脊柱脊髓杂志,2000,10：15-17.
[13]Rannou F,Corvol MT,Hudry C,et al.Sensitivity of anulus fibrosus cells to interleukin 1 beta.Comparison with articularchondrocytes[J].Spine,2000,25(1): 17—23.
[14]龚非力.医学免疫学第二版.北京：科学出版社,2004,6.
[15]Pelletier JP, Dibattishta JA, Rouyhley P,et al. R beum Dis, Clin North(Am),1993, 19(3):545～568.
[16][Kang JD,Stefanovic(Racic M,McIntyre LA,et al.Toward a biochemical understanding of human intervertebral disc degeneration and herniation: Contributions of nitric oxide, interleukins,prostaglandin E2,and matrix metalloproteinases[J].Spine,1997,22:1065-1073.
[17]李新友,王民,刘淼,等.白细胞介素-6在突出的腰椎间盘中的表达及其意义.中国脊柱脊髓杂志,2001,8：581-582.
[18]Burke JG, G Watson RW, Conhyea D, et al. Human Nucleus Pulposis Can Respond to a Pro-inflammatory Stimulus[J]. Spine,2003,28(24):2685-269.
[19][赵太茂,刘森,宋红星,等.突出椎间盘组织中TNF-a与IL-b的表达及意义[J].中国脊柱脊髓杂志,1999,9(1)：17.
[20]Valdes,AnaM,Hassett, Geraldine MBBS. Radiographic Progression of Lumbar Spine Disc Degeneration Is Influenced by Variation at Inflammatory Genes:A Candidate SNPAssociation Study in the Chingford Cohort[J]. spine,2005,30(21): 2445.
[21]Rand N,Reichert F,Floman Y,et al.Murine nucleus pulposus-derived cells secrete interleukins-1-beta,-6,and-10 and granulocyte-macrophage colony-stimulating factor in cell culture.Spine 1997;22(22):2598-2601.
[22]Kato T,Haro H,Komori H,et al.Sequential dynamics of inflammatory cytokine,angiogenesis inducing factor and matrix degrading enzymes during spontaneous resorption of the herniated disc[J].J Orthop Res,2004,22(4):895—900.
[23]YamanakaH, MakinoK, TakizawaM, et al.Expression and tissue localization of membrane-types 1,2, and 3 matrixmetalloproteinases in rheumatoid synovium. Lab Invest,2000,80:677-687.
[24]Hammacher S,Matern S,Rroeb E.Extracellular matrix-form basic research to clinical significance:an overview with special consideration of metalloproteinases J.Dtsch Med Wochenschr-.2004,129-(38):1976-1980.
[25]Massova I, Kotra LP, Fridman R, eta.l. Matrixmetalloproteinases:structures, evolution, and diversification. FASEB J,1998,12:1075-1095.
[26]Gardner J,Ghorpade A.Tissue inhibitor of metalloproteinase (TIMP)-1:the TIMPed balance of matrix metalloproteinases in the central nervous system[J].J Neurosci Res,2003,74(6):801-806.
[27]Weiler C,Nerlich AG,Zipperer J,et al.2002 SSE Award Competition in Basic Science:expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorption[J].Eur Spine J,2002,11(4):308-320.
[28]Roberts S,Caterson B,Menage J,et al.Matrix metalloproteinases and aggrecanase:their role in disorders of the human intervertebral disc [J]. Spine, 2000,25(23):3005-3013.
[29]Kanemoto M,Hukuda S,Komiya Y,et al.Immunohistochemical study of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 human intervertebral discs[J].Spine,1996,21(1):1-8.
[30]Semenza GL, Wang GL·A nuclear factor induced by hy-poxia via denovo protein synthesis binds to the human erythropoietin gene enhancer at a site required for tran-scriptional activation-Mol Cell Bio,l 1992,12:5447-5454.
[31]Zheng JZ, Leung SW, Weisz A, et al·Transactivation and inhibitory domains of hypoxia-inducible factor lalpha modulation of transcriptional activity by oxygen tension·J BiolChem,1997,272 (31):19253-19260.
[32]Pugh CW, Ratclife PJ·Regulation of angiogenesis by hy-poxia:role of theHIF system-NatMed,2003,9:677-68.
[33]王俊丰,贾长青,刘洪亮等.缺氧诱导因子-1α和血管内皮生长因子在突出腰椎间盘中的表达及意义[J].中国组织化学与细胞化学杂志,2008,17(1)：65-69.
[34]Liu GZ,Ishihara H,Osada R,et al.Nitric oxide mediates the change of proteoglycan synthesis in the human lumbar intervertebral disc in response to hydrostatic pressure[J]. Spine,2001,26:134-141.
[35]Kohyama K,Saura R,Doita M, et al. Intervertebral disc cell apoptosis by nitric oxide:Biological understanding of intervertebral disc degeneration[J].Kobe J Med Sci,2000,46:283-295.
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