正常和病变椎间盘髓核的糖胺多糖研究

蛋白多糖是椎间盘髓核的主要基质成分 ,为研究蛋白多糖代谢变化与椎间盘退变、突出的关系 ,我们对椎间盘髓核中蛋白多糖的水解提取物———糖胺多糖 (Ｇｌｙｃｏｓａｍｉｎｏｇｌｙｃａｎ ,ＧＡＧ)进行了生化研究。1　材料与方法1 1　取材 :正常椎间盘髓核标本取自急性脑外伤死后 1ｈ的男性青年 (平均年龄 2 6 9岁 )共 34个标本 ,肉眼及解剖观察无明显变性和椎间盘突出。退变标本取自腰椎间盘突出症手术患者 ,共 33个 ,患者平均年龄 2 8岁。1 2　ＧＡＧ提取 :将标本称取重量后切碎 ,放人编号试管中 ,依顺序加入0 1ＭＮａＯＨ1 5ｍｌ,制成…     

椎间盘纤维环中糖胺多糖的实验研究

目的　研究正常和退变腰椎间盘纤维环中糖胺多糖 (GAG)含量及其主要成份百分组成 ,探讨椎间盘退变的发生机理。方法　对 34个正常纤维环和 35个椎间盘突出症纤维环标本中GAG含量进行测定 ,并用醋酸纤维素膜电泳和光密度扫描的方法对GAG主要成份百分组成进行测定。结果　正常纤维环GAG含量为 4 17± 0 94g/10 0 g ,退变纤维环GAG含量为 3 5 7± 0 98g/10 0g ,低于正常组 (P <0 0 5 ) ;退变组GAG中硫酸软骨素 (CS)百分值低于正常组 (P <0 0 5 ) ;而其透明质酸 (HA)和硫酸角质 (KS)高于正常组 (P <0 0 5 )。结论　椎间盘纤维环中GAG含量下降 ,是引起纤维环胶原纤维稳定性降低 ,影响纤维环板层滑动 ,导致椎间盘退变、突出症发生的重要原因之一。退变纤维环GAG含量下降由其主要成分CS含量下降所致     

脊柱侧凸顶椎区椎间盘纤维环中多聚蛋白多糖的表达及意义

目的：探讨脊柱侧凸患者顶椎区椎间盘纤维环中多聚蛋白多糖的表达及其意义。方法：采集2003年7月至2004年9月间行前路松解或矫形手术的40例脊柱侧凸患者侧凸顶椎区椎间盘组织。青少年特发性脊柱侧凸患者（adolescent idiopathic scoliosis，AIS）25例，胸椎椎间盘11例，腰椎椎间盘14例；先天性脊柱侧凸患者（congenital scoliosis，CS）15例，胸椎椎间盘6例，腰椎椎间盘9例。利用RT—PCR扩增多聚蛋白多糖（Aguecan），琼脂糖凝胶电泳，在UVP（紫外光测定法）成像系统进行扫描．GelWork图像分析系统中进行灰度测定半定量分析。分别计算AIS组和CS组凹侧、凸侧纤维环中Aggrecan含量，并对CS组和AIS组胸椎和腰椎以及椎间盘的凹侧和凸侧进行比较。结果：AIS组和CS组椎间盘凹侧纤维环中Aggrecan含量低于凸侧．差异有显著性（P〈0．01），胸椎椎间盘纤维环中Aggrecan的含量低于腰椎，但无统计学差异。AIS组Aggrecan的含量和CS组相应部位Aggrecan的含量无明显差别。结论：AIS椎间盘纤维环凹凸侧存在Aggrecan代谢差异．并且可能是脊柱侧凸所致的继发改变．但也可能是脊柱侧凸发生、发展中的重要因素。     

腰间盘髓核中胶原及蛋白多糖含量的测定

测定了５０例正常腰椎间盘髓核及５０例腰椎间盘突出症髓核中胶原及蛋白多糖含量，确立了国人腰间盘髓核胶原及蛋白多糖含量的正常值范围，为研究病态腰间盘髓核胶原及蛋白多糖的代谢提供了正常对照参数。通过对腰间盘突出症髓核胶原和蛋白多糖的定量分析及与正常比较，观察到突出髓核中胶原含量明显高于正常而蛋白多糖含量明显降低（Ｐ值＜０．００１），从而进一步证实了腰间盘突出症的病理变化，并从生物化学及生物力学角度进行了分析。     

颈椎动力平衡失调大鼠椎间盘蛋白多糖的观察

目的探明颈椎动力平衡失调对颈椎间盘退变的影响乃至在颈椎病发病机制中的作用。方法选择了２０只ＳＤ大鼠,通过手术直接损伤其颈背部浅层、深层及全层肌群,建立了大鼠颈椎动力平衡失调程度不同的动物模型,６月后观察各造模组颈椎间盘内蛋白多糖量的变化,并与对照组相比较。结果大鼠颈椎动力平衡失调可影响颈椎间盘内蛋白多糖的代谢,加速其降低,依颈椎动力平衡失调程度不同而有差异,影响程度由轻至重依次为深层、浅层、全层肌群损伤组。结论颈椎动力平衡失调可加速颈椎间盘退变,并在颈椎病发病机制及防治中意义重大     

颈椎间盘压缩力学性能改变与终板蛋白多糖变化关系的实验研究

目的:探讨颈椎间盘退变时基质各成分的变化及其对椎间盘力学性能的影响。方法:将24只家兔随机分为对照组和模型组(n=12),模型组保持45°低屈曲位5h/次/d,最长持续3个月。分别于造模后1、2、3个月时各处死4只动物,取C5/6椎间盘测定终板糖胺多糖(glycosaminoglycan,GAG)总量、硫酸软骨素(chongdroitinsulphate,CS)与硫酸角质素(keratansulphate,KS)比值和透明质酸(hyaluronicacid,HA)含量;同时作力学测定。结果:模型组终板抗压强度、断裂时的最大形变、终板基质GAG总含量、CS/KS比值、HA含量都低于对照组(P<0.05),并随造模时间的延长两组差异越显著。结论:长时间的应力不均可造成椎间盘终板材料力学特性改变;终板蛋白多糖含量和成分改变可能是颈椎间盘生物力学性能减退的主要原因之一。     

CDMP-1逆转人退变椎间盘髓核细胞的实验研究

[目的]通过CDMP-1促进退变髓核细胞外基质合成来逆转椎间盘退变.[方法]分离、培养人退变椎间盘髓核细胞,取第3代髓核细胞,应用CDMP-l分组进行干预实验.A组为对照组,B组和C组为实验组,B组加人100ng/ml CDMP-1,C组加入200ng/rnl CDMP-1.采用光学相差显微镜及透射电镜对对照组和实验组细胞形态和超微结构进行对比观察:XTT-PMS法检测各组髓核细胞12d的生长曲线,研究三组细胞间的生长动力学差异;实时荧光定量PCR检测髓核细胞,7,14,21d Ⅱ型胶原和糖胺多糖的mRNA表达.[结果]光学相差显微镜及透射电镜示:两剂量实验组形态学上没有明显差异,与对照组相比,CDMP-1实验组能较好的维持髓核细胞生物形态学特性,延长退变髓核细胞的衰老;三实验组12d的生长曲线一致,两剂量的CDMP-1没有增加退变髓核细胞的增殖;实时荧光定量PCR示100ng/ml CDMP-1组:Ⅱ型胶原mRNA表达总体均数及3个时间点均高于另外两组任一时间点(P<0.05);糖胺多糖mRNA表达总体均数及14、21d 2个时间点均高于另外两组相应时间点(P<0.05),且Ⅱ型胶原和糖胺多糖mRNA表达量随刺激时间增加而增加.200 ng/ml CDMP-1组:提高了糖胺多糖mRNA表达水平的同时,却降低了Ⅱ型胶原mRNA水平.[结论]100ng/ml的CDMP-l可以延长体外培养人退变髓核细胞的衰老,维持细胞生物形态学特性,提高退变髓核细胞Ⅱ型胶原和糖胺多糖mRNA表达水平,在一定程度逆转了椎间盘髓核细胞退变.     

人腰椎间盘蛋白多糖聚合体分布的研究

目的：以Ｒｈｏｄａｍｉｎｅ标记的核心蛋白作为荧光探针，观察腰椎间盘中的蛋白多糖聚合体的分布。方法：将小牛的肩胛软骨溶于含蛋白酶抑制剂的４．０Ｍ盐酸胍中，通过分解状态下的高速离心、透析、聚合状态下的高速离心提取出蛋白多糖聚合体。然后经分解状态下的超速离心、酶解、层析及与荧光素耦合等方法制备出一种带有荧光素的核心蛋白，作为透明质酸的特异性荧光探针来观察椎间盘中蛋白多糖聚合体的分布。结果：胎儿、正常成年     

凝固椎间盘髓核的生物力学研究

目的观察凝固髓核的轴向抗压缩能力,探讨其临床应用的可行性。方法将12只杂种犬的16个椎间盘分为4组：空白对照组4个,明矾溶液1组（注入明矾溶液3d）4个,明矾溶液2组（注入明矾溶液2周）4个,明矾溶液3组（注入明矾溶液1个月）4个。外接压力泵,并维持160kPa压力5min,将0．15ml10％明矾溶液缓慢注入椎间盘髓核,使椎间盘髓核凝固以实验椎间盘为中心,取一个脊柱功能单位作为生物力学检测的标本。检测标本的轴向载荷位移曲线及使标本髓核突出的轴向载荷。结果轴向载荷500N时,明矾溶液凝固的髓核（3个时间组）与正常椎间盘被压缩的位移无统计学差异;轴向载荷2058N（210kg）时,正常椎间盘髓核可被挤出;轴向载荷2538N（259kg）时,明矾溶液凝固的髓核（1个月）被挤出。结论明矾溶液可使椎间盘髓核产生凝固,凝固髓核的轴向抗压能力略有增强,可达到强化髓核的目的。     

颈椎间盘纤维环及髓核的超微结构观察

目的：探讨在颈椎间盘纤维环及髓核退变中组织形态的变化。方法：对正常人、单纯颈椎间盘突出症、脊髓型颈椎病三组椎间盘纤维环及髓核进行电镜观察。结果：三组椎间盘胶原纤维无明显变化,单纯颈椎间盘突出症与脊髓型颈椎病人的退变椎间盘细胞较正常人有明显变化,表现为严重退变或细胞坏死。结论：单纯颈椎间盘突出症患者椎间盘以退变细胞为主,为退变早期阶段功能代偿期,脊髓型颈椎病椎间盘以坏死细胞为主,为退变晚期阶段,为不可逆期;颈腰椎间盘退变的组织形态学不完全相同。     

腰椎间盘前缘与后缘高信号区的临床对照研究

目的 通过与腰椎间盘后缘高信号区(HIZ)比较,探讨前缘HIZ的发生规律、分布及临床意义.方法 根据HIZ的诊断及定位,将610例有完整临床资料的腰椎MRI,依据有无HE及其发生部位分为对照组(无HIZ),前缘组,后缘组和前后缘组.统计总样本中HIZ发生率,对比分析各组性别比例、年龄、体重、腰痛发生率及HIZ分布规律.结果 610例患者中,对照组315例(51.6%),前缘组95例(15.6%,119个椎间盘),后缘组159例(26.1%,189个椎间盘),前后缘组41例(6.7%,96个椎间盘).前、后缘组发生率间存在显著的统计学差异(P<0.01).各组的性别构成和体重载明显差异(P>0.05),年龄间存在显著的统计学差异(P<0.01,对照组<后缘组<前后缘组<前缘组).前缘组多见于L1,2～L4,5,后缘多位于L3,4～L5S1对照组、前缘组、后缘组和前后缘组腰痛发生率分别为40.0%,52.6%,55.4%和65.8%,对照组明显低于其他三组(P<0.05),后三组间差异无统计学意义(P>0.05).结论 与纤维环后缘HIZ相比,前缘HIZ发生率较低,患者年龄较大,发生节段较高,与腰痛症状亦明显相关.     

Effects of axial traction stress on solute transport and proteoglycan synthesis in the porcine intervertebral disc in vitro

Summary The effects of axial traction stress on intradiscal hydration, solute transport and proteoglycan synthesis were examined in 658 porcine coccygeal intervertebral discs in vitro. Measurements were performed in three tissue fractions: nucleus pulposus, inner and outer annulus fibrosus. At 0.80 MPa traction stress, the equilibrium hydration did not change in the nucleus pulposus. However, in the inner and outer annulus, the equilibrium hydration was reduced, and the change led to an increase of the effective fixed charge density. Diffusion of solute to the nucleus pulposus was significantly suppressed at 0.80 MPa traction stress. The fluid flow of the intervertebral disc tended to be suppressed during the creep recovery process after compression. The proteoglycan synthesis rate in the outer annulus was markedly suppressed by traction stress of 0.80 MPa for 4 h, but not that in the nucleus pulposus. These results suggest that a prolonged excessive axial traction stress induces a decrease in tissue hydration in the annulus fibrosus, and this may lead to an increase in the fractional volume of solid in the matrix and tissue osmotic pressure, resulting in diffusion inhibition of solute and suppression of proteoglycan synthesis. Thus, prolonged and excessive spinal traction may accelerate disc degeneration.Presented at the annual meeting of the International Society for the Study of the Lumbar Spine, May 12–16, 1991, Heidelberg, Germany     

白细胞介素-6对椎间盘细胞中蛋白多糖代谢影响的实验研究

目的:研究白细胞介素-6(interleukin-6,IL-6)对椎间盘纤维环和髓核细胞中蛋白多糖代谢的影响。方法:自然流产的胎儿4例,4h内无菌取出椎间盘,分别进行纤维环和髓核细胞的体外培养。在纤维环细胞的培养液中分别加入IL-60(对照组)、400、800ng/ml,培养24h后,用Alcian法检测培养液中硫酸软骨素的含量。在培养中的髓核细胞中加入IL-60(对照组)、100、400、800ng/ml,培养24h,然后测量培养液中硫酸软骨素的含量。结果:在纤维环细胞中加入IL-6组较不加IL-6对照组培养液中硫酸软骨素的含量增加,在髓核细胞组中加入IL-6组和对照组的差别无显著性。结论:IL-6可以刺激椎间盘纤维环细胞中蛋白多糖的合成,但对髓核细胞中蛋白多糖的合成没有明显的作用。     

腰椎间盘突出症非手术治疗规范化的思考与建议

腰椎间盘突出症为坐骨神经痛的常见病因，多种非手术治疗疗效满意，并在多国被推荐使用。非手术疗法的有效性与优势取决于如何被有效运用。为此，该文提出医患双方的积极态度、正规治疗方案与足够的治疗时间，以及多种疗法的程式化运用是规范该病非手术治疗的基本要求。     

一氧化氮在静水压下对人体腰椎间盘蛋白多糖合成的影响

[目的]探讨一氧化氮对人体腰椎间盘组织蛋白多糖合成的影响及静水压对蛋白多糖合成调节过程中一氧化氮是否起到中间介质作用。[方法]72例后方腰椎间盘摘除术的志愿患者中获取新鲜的72个椎间盘样品被切成1～2mm^3细片后，与1ml培养基DMEM一同装入塑料注射器中，放入培养椎间盘组织的压力装置，蛋白多糖合成由^35S-硫酸盐结合率来测定。无活性、稳定的一氧化氮的最终产物亚硝酸盐浓度由分光光度法来测定。[结果]静水压在0．3MPa蛋白多糖合成最高，约为0．1MPa的1．3倍；3MPa抑制蛋白多糖合成率。但静水压对一氧化氮的产生起着逆转效应。在0．3MPa一氧化氮的产生量和0．1MPa的相比略微降低，而3MPa增加一氧化氮的产生量，约是0．1MPa的1．34倍。一氧化氮合成酶的竞争性抑制剂N^C-methyl-L-arginine（简称L-NMA，10-1000μmol）在0．3MPa诱导增加蛋白多糖合成率，而3MPa降低对蛋白多糖合成率的抑制作用。[结论]在人体腰椎间盘细胞静水压影响着一氧化氮生成，且一氧化氮对蛋白多糖合成率改变起着中间介质调节作用。     

The anabolic effect of plasma-mediated ablation on the intervertebral disc: stimulation of proteoglycan and interleukin-8 production

Background context

Plasma-mediated radiofrequency-based ablation (coblation) is an electrosurgical technique currently used for tissue removal in a wide range of surgical applications, including lumbar microdiscectomy. In vitro and in vivo studies have shown the technique to alter the expression of inflammatory cytokines in the disc, increasing the levels of interleukin-8 (IL-8), which may promote maturation and remodeling of the disc matrix.

Purpose

To better understand the effect of coblation treatment, this study characterizes the temporal and spatial pattern of healing after stab injury to the rabbit intervertebral disc, with and without plasma-mediated radiofrequency treatment.

Patient sample

A total of 23 New Zealand white rabbits.

Study design

Annular and nuclear stab injuries.

Outcome measures

Sandwich enzyme-linked immunosorbent assay evaluated the concentrations of cytokines tumor necrosis factor-α, IL-1β, and IL-8. Histopathologic evaluations were performed on whole discs and end plates. Tissue sections were stained with Safranin-O to evaluate nucleus pulposus and annulus fibrosus proteoglycan content and with Alcian blue for extracellular proteoglycan content. Intradiscal leakage pressure was evaluated by injecting methylene blue dye into the nucleus.

Methods

Animals underwent annular and nuclear stab injuries on three consecutive lumbar discs (L2–L3 to L4–L5). The three levels were randomly assigned into one of the three groups for treatment with a plasma-mediated radiofrequency ablation device (TOPAZ; ArthroCare Corp., Austin, TX, USA): active treatment of the nucleus only (SN); active treatment of both nucleus and annulus (SNA); sham treatment. Unstabbed/untreated discs from L5–L6 (n=5) served as normal controls. Animals were euthanized at 4, 8, and 28 days postsurgery.

Results

Tumor necrosis factor-α was detected in sham discs at 4 and 8 days, but not in coblation groups (SN or SNA); IL-1β was below detection in all three treatment groups. Interleukin-8 levels increased in all treatment groups at 4 and 8 days compared with normal control, peaking at 4th day for sham and SN groups and 8th day (p>.3) for the SNA group (a 2.5-fold increase). Pressure measurements revealed higher leakage in the SN group, but no statistically significant differences. Histopathology showed higher proteoglycan production by 28 days in the SNA and SN groups compared with sham. All three treatment groups showed ruptured annular fibers from the stab injury, but maintained the overall architecture. Remnants of notochordal tissue within the nucleus were evident in all treatment groups at 4 and 8 days, but were only found in sham group by 28 days. At this time, unlike the normal or sham controls, the nucleus of SN and SNA discs had fibrocartilaginous tissue with chondrocyte-like cells. Significant differences in the disc architecture grade were only noted when comparing normal controls with other groups by 28 days (p<.001).

Conclusions

Plasma-mediated radiofrequency ablation appears to have an anabolic effect on disc cells, stimulating proteoglycan and IL-8 production and maintaining annulus architecture. Coblation treatment appears to reduce cellular response to proinflammatory stimuli and restore overall disc architecture that may prove beneficial in a number of degenerative disc paradigms. Further studies are encouraged to investigate the therapeutic effect of the technique.     

Effect of age on the abundance and fragmentation of link protein of the human intervertebral disc

The link proteins of the human intervertebral disc were studied in tissue extracts by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), followed by immunoblotting, using a specific monoclonal antibody. Three link proteins were detected, corresponding in electrophoretic mobility to those present in articular cartilage. As with articular cartilage, the largest link protein predominates in the young, whereas in the adult the smallest link protein is equally abundant and internal fragmentation of the link proteins occurs. Only in the newborn is the quantity of extractable link protein comparable to that from articular cartilage. In the adult, the disc contains much less link protein than is present in autologous articular cartilage. Neither the amount nor heterogeneity of the link protein differs among different levels within the lumbar spine, although the proportions of the three proteins can differ between the anulus fibrosus and nucleus pulposus. The anulus always contained more extractable link protein relative to tissue wet weight than the nucleus, and the nuclear link protein, at least in adolescents, contained a greater proportion of the smallest link protein. Such changes in the quantity and structure of the disc link proteins may affect the properties of the proteoglycan aggregates and, thus, could influence disc function.     

The influence of scoliosis and ageing on proteoglycan heterogeneity in the human intervertebral disc

Proteoglycans (PGs) purified from 19-year-old male (19M), 53-year-old female (53F), and 15-16-year-old adolescent idiopathic scoliotic (AIS) disc tissues were characterised by their aggregation properties with hyaluronic acid (HA), glycosaminoglycan (GAG), and amino acid composition, electrophoretic mobilities by composite agarose polyacrylamide gel electrophoresis (CAPGE), and hydrodynamic size by Sepharose CL2B gel permeation chromatography. Proteoglycan levels were similar in the AIS and 53F discs, but considerably higher levels were found in the 19M discs. Proteoglycan populations assessed by the aforementioned criteria demonstrated a similar level of heterogeneity in the AIS and 53F discs but this differed with the 19M sample. Two small Mr, low buoyant density PG species were particularly prominent in AIS and 53F discs: these were similar to two nonaggregating PG species formerly reported by di Fabio et al.; however, the corresponding PG species in 19M discs were of somewhat different composition. Collectively, these data suggest similar processing of PGs in the AIS and 53F discs. Furthermore, the pattern of normal age changes in the PGs from AIS discs appears to be disturbed, most likely arising as a consequence of the abnormal biomechanical forces intrinsic to the deformity.     

脊柱节段血管阻断对椎间盘退变发生发展的影响

目的：探讨结扎脊柱节段血管对椎间盘退变发生发展的影响。方法：16只成年犬随机平均分为两组．每只犬均结扎左侧T6～T9的节段血管。分别于造模后3个月和8个月急性失血法各处死一组动物，处死后立即取整段胸椎。T6，7至T8／9为实验区(结扎节段血管区)，取结扎血管以上的T4／5椎间盘为病理检查中的对照区，T6／7为病理检查实验区；T7／8和F8/9椎间盘为生化分析的实验区，结扎血管以下的T11/12、T12／13椎间盘为生化分析的对照区。对对照区和实验区内椎间盘的形态和成分进行比较。结果：与对照组相比．实验组的椎间盘在3个月时胶原成分已有显著增加，而糖胺多糖成分显著降低；8个月组与3个月组无显著性区别。结论：在脊柱节段血管阻断后，椎间盘的周围血供减少，影响椎间盘的营养供应，可诱发或加速椎间盘的退变。