椎间盘源性下腰痛与椎间盘内神经生长

疼痛的椎间盘外层纤维环、内层纤维环和髓核中都有神经纤维分布。体外研究发现椎间盘基质成分中蛋白多糖含量和结构变化,尤其是硫酸软骨素含量变化对神经生长具有明显的调节作用。在退变的椎间盘中,硫酸软骨素含量减少有利于椎间盘外周分布的神经纤维向内层纤维环甚至髓核内生长,从而导致椎间盘源性下腰痛。     

BMP-2对人退变椎间盘髓核细胞影响的实验研究

[目的]探讨BMP -2对人退变髓核细胞合成细胞外基质的影响.[方法]分离、培养人退变椎间盘髓核细胞,取第2代髓核细胞,随机将退变推间盘髓核细胞分为2组.A组:加入100 ng/ml BMP -2,B组:加入200 ng/mlBMP -2,C组:对照组,不加干扰因素.通过对试验组和对照组髓核细胞采用光镜、电镜等形态学方法进行大体形态和超微结构观察,细胞Ⅱ型胶原和糖胺多糖的mRNA表达.ELISA检测细胞培养上清中人Ⅱ型胶原含量,DMMB比色法检测细胞培养上清中糖胺多糖含量.[结果]髓核细胞中Ⅱ型胶原、糖胺多糖表达水平实验组均高于对照组.[结论] BMP-2蛋白可促进退变腰椎间盘细胞分泌蛋白多糖和Ⅱ型胶原,增加细胞活性,恢复椎间盘的功能和活性,因此运用BMP-2椎间盘内注射有望成为椎间盘退变疾病生物治疗的方法之一.     

白细胞介素-1受体拮抗剂对兔椎间盘髓核前列腺素和5-羟色胺代谢的影响

目的:研究白细胞介素-1受体拮抗剂(IL-1Ra)对兔椎间盘髓核前列腺素E2(PGE2)、前列腺素F1琢(PGF1琢)和5-羟色胺(5-HT)代谢的影响。方法:取日本大白兔椎间盘髓核,制成匀浆后进行髓核组织的体外培养,观察不同浓度IL-1Ra和同一浓度IL-1Ra不同培养时间培养液中PGE2、PGF1琢和5-HT的含量。结果:IL-1Ra(100、200、400ng/ml)实验组较对照组培养液中PGE2、PGF1琢和5-HT的含量显著减少(P<0.01),呈现明显的浓度依赖性;IL-1琢(2.0ng/ml)组和IL-1Ra(200ng/ml)组随培养时间延长,PGE2、PGF1琢和5-HT的含量增加,每个时间段实验组与对照组浓度之差的差异有显著性意义(P<0.01)。结论:IL-1Ra可以拮抗IL-1琢对椎间盘髓核细胞PGE2、PGF1琢和5-HT合成的促进作用,呈现明显的浓度依赖性。IL-1Ra可以较长时间拮抗IL-1琢生物学作用,但本实验未能证明此作用具有时间依赖性。     

中性蛋白酶引起人体椎间盘退变的研究与分析

人体椎间盘由髓核、纤维环及透明软骨板组成。髓核基质及纤维环主要由蛋白多糖及胶原组成。研究表明在椎间盘退变过程中,蛋白多糖含量下降而硫酸角蛋白浓度升高。由此推断椎间盘退变可能是由于基质合成与分解不平衡所致。Melrose及Sedowofia等陆续报道从人体椎间盘中提取出具有活性的胶原蛋白溶解酶和弹性蛋白溶解酶,从而揭示并证实在椎间     

微载体立体培养腰椎间盘细胞蛋白多糖表达的研究

目的研究腰椎间盘细胞在微载体培养与单层培养中细胞表达蛋白多糖含量的差别。方法椎间盘疾病手术病例的术中切除组织采用酶消化法分别进行微载体三维细胞培养和单层细胞培养；取胎儿椎间盘组织，显微镜下区分髓核细胞和纤维环细胞，分别进行培养，同成入组对照。利用^35S放射标记渗入放免定量测定的方法进行蛋白多糖含量的检测。结果①椎间盘细胞胞内的蛋白多糖含量（cpm），细胞单层培养组为101．909±11．439，微载体立体培养组为136．607±10．792，P〈0．05；②椎间盘细胞表达的蛋白多糖含量（cpm），细胞单层培养组为105．119±13．040，微载体立体培养组为174．231±17．676，P〈0．05；③各组椎间盘细胞表达的蛋白多糖含量均高于细胞内的含量；④胎儿腰椎间盘细胞蛋白多糖的含量及表达量均高于成人退变椎间盘细胞，胎儿髓核细胞蛋白多糖的表达量高于纤维环细胞的表达量。结论椎间盘细胞的微载体三维立体培养相对单层培养具有较高细胞蛋白多糖的表达量，是一种较好的细胞培养方式。     

IL-1α对体外培养兔椎间盘髓核组织PGE2、PGF1α及5-HT代谢的影响

目的 研究自细胞介素1α(IL-1α)对体外培养的兔椎间盘髓核组织前列腺素E2(PGE2)、前列腺素F1α(PGF1α)和5-羟色胺(5-HT)代谢的影响。方法 取日本大白兔，无菌手术摘取椎间盘髓核，制成匀浆后进行髓核组织的体外培养。实验组加入IL-1α，对照组空白，培养12、24、30、72h，分别测定各时段培养液中PGE2、PGF1α、5-HT的浓度。结果 各时段实验组和对照组的差异均有显著性，随着培养时间的延长，实验组培养液中PGE2、PGF1α、5-HT的含量均逐步增高，差异有显著性。结论 IL-1α刺激体外培养的兔椎间盘髓核细胞合成PGE2、PGF1α和5-HT，呈现明显的时间依赖性。     

实验性脊柱内固定后相应区域椎间盘超微结构观察

目的　观察脊柱内固定后相应区域椎间盘的超微结构变化。　方法　日本大耳白兔2 4只,随机分成实验组和对照组,每组12只。实验组骨膜下游离T1 0 ～L3棘突和关节突,克氏针制成“L”形,将钢丝横行穿过T1 1、1 2 ,L1、2 的关节突关节,并与置于T1 1 ～L3棘突两旁的克氏针系紧,对相应区域的脊柱行内固定术。对照组未行手术,仅喂养至实验完成。术后6个月,对两组动物摄X线片观察1次,随后处死动物。取两组动物的L1 椎间盘组织(髓核、纤维环内侧及纤维环外侧)行透射电镜观察,对两组T1 2 、L2 椎间盘组织分别行水平面和矢状面透射电镜及扫描电镜观察。　结果　X线片显示,实验组与对照组椎体及椎间隙差别不明显;透射电镜与扫描电镜观察,实验组椎间盘的髓核、纤维环内层细胞的结构改变较纤维环外层早;对照组的髓核、纤维环内层细胞的结构改变与纤维环外层差别不明显。在退变的椎间盘基质中,蛋白多糖颗粒和特殊结构明显减少。髓核与纤维环基质内有蛋白多糖颗粒和一种特殊结构,而特殊结构在髓核与纤维环内层的形态不一致。　结论　脊柱内固定术后6个月,实验组在异常应力环境下发生椎间盘退变。髓核、纤维环内层基质内的特殊结构分布有特殊规律,与蛋白多糖颗粒在椎间盘退变中的生物学行为密切相关。     

影响椎间盘退变生物化学改变的因素

椎间盘退卞的生物化学改变表现为 :蛋白多糖含量减少 ,硫酸角质素与硫酸软骨素的比例增加 ,Ⅰ型型胶原增多 ,Ⅱ型胶原减少 ,水分减少等 ,这一变化严重影响了椎间盘的生物力学特性 ,由此构成了椎间盘突出的病理基础。近年来 ,随着分子生物学、分子免疫学等医学基础学科的迅速发展 ,椎间盘退变生物化学改变及其影响因素等方面的研究取得了一些进展 ,现在综述如下 :1　应力对椎间盘退变的生物化学影响椎间盘由四周的纤维环、中心的髓核及上下软骨终板组成。正常椎间盘髓核呈流体静压状态 ,椎间盘内压为轴间压载荷的 1.3～ 1.5倍 ,当外载荷达 2 …     

芹菜素在静水压下对人体腰椎间盘髓核蛋白多糖核心蛋白基因表达的影响

[目的]探讨中药提取物芹菜素在静水压下对体外培养的人体腰椎间盘髓核蛋白多糖核心蛋白基因表达的影响.[方法]32例后路腰椎间盘髓核摘除术的志愿患者中获取的32个新鲜椎间盘髓核样品,每1例髓核样品被切成1～2 mm3大小的碎块,与1 ml培养基DMEM一同装入2.5 ml的注射器中.培养基中分别加入芹菜素(Apigenin)、一氧化氮合成酶的竞争性抑制剂NG-Monomethyl-L-arginine(简称L-NMMA)及一氧化氮的供体Sodium Nitroprusside(简称SNAP),在静水压装置中培养2 h后,对每组中的髓核样品进行石蜡包埋、切片,通过原位杂交来研究芹菜素对椎间盘髓核蛋白多糖核心蛋白的基因转录水平的调节作用.[结果]芹菜素3 atm组和L-NMMA 3 atm组蛋白多糖核心蛋白原位杂交结果显示阳性髓核细胞数最多,平均光密度值(MOD)最大.[结论]对于退变的椎间盘的髓核组织,芹菜素能够促进蛋白多糖核心蛋白mRNA的基因转录水平.     

聚集蛋白聚糖酶-1在退变椎间盘中的表达及意义

[目的]探讨聚集蛋白聚糖酶-1(aggrecannase-1,Agase-1)在退变椎间盘组织中的表达、规律及其意义。[方法]实验组为手术切除的腰椎间盘突出患者的退变椎间盘组织,分为纤维环破裂组和未破裂组;对照组为取自腰椎骨折患者的正常腰椎间盘组织。应用HE染色检测各组椎间盘组织的病理形态学变化,RT-PCR检测Agase-1mRNA的表达;Western印迹法检测蛋白多糖(aggrecan)的含量。[结果]Agase-1mRNA在实验组中表达均明显高于对照组,纤维环破裂组明显高于纤维环未破裂组(P<0.01),Aggrecan在实验组中的含量低于对照组,纤维环破裂组中Aggrecan明显低于纤维环未破裂组(P<0.01),HE染色显示髓核细胞数量随着椎间盘退变程度的增加显著减少,纤维环破裂组髓核组织中有血管生成,伴有炎细胞浸润。[结论]Agase-1可能参与了人类椎间盘组织的退变过程,且在突出的椎间盘组织中有增高趋势。     

A study on topographical change of proteoglycans in human lumbar disc

Proteoglycan aggregates, the binding of disc proteoglycan to hyaluronate, chondroitin sulfate chain length, hexosamine and amino acid compositions were measured in different regions of the L1/L2 and L4/L5 intervertebral discs from 3 spines aged 35, 49, and 60 years. The results were as follows: proteoglycan aggregates and the binding of disc proteoglycan to hyaluronate were shown to increase from the nucleus pulposus to the outer annulus, while only small differences were observed among the anterior, posterior and lateral regions in either the inner or the outer annulus within a given disc. Changes in chondroitin sulfate chain length were observed with aging. However, within a given disc, and furthermore, within a given spine, chondroitin sulfate chains were similar in length. Glucosamine/galactosamine molar ratios of disc proteoglycan were higher in the L1/L2 disc than the L4/L5 disc. Changes with age were also observed. Nevertheless, only minor differences among the anterior, posterior and lateral regions of the annulus fibrosus were found within a given disc. The amino acid compositions showed small differences when proteoglycans from various regions of a disc were compared. All of them had a high content of aspartic acid, threonine, serine, glutamic acid, proline and glycine. The results indicate that it seems there are only minor differences among the anterior, posterior and lateral regions of the annulus fibrosus in a number of the experimented parameters.     

白细胞介素—6在突出的腰椎间盘中的表达及其意义

目的对白细胞介素-6(IL-6)在突出的腰椎间盘组织中的含量及产生IL-6的组织细胞类型进行研究,并对其意义进行探讨.方法对12例正常及突出腰的椎间盘组织进行体外培养,用放免方法测定培养液上清中IL-6含量;并在20例突出的腰椎间盘组织中用免疫组化方法对产生IL-6的细胞类型及组织学定位进行研究.结果突出的腰椎间盘组织产生IL-6的量为(987.53±594.44)pg/g,正常对照组IL-6的量为(114.21±63.91)pg/g,两者统计学有显著性差异(P＜0.001),免疫组化结果显示白细胞介素-6阳性细胞在突出椎间盘周围的肉芽组织中表达最强,主要以成纤维细胞、淋巴细胞及软骨细胞为主.结论腰椎间盘组织可自身合成IL-6,IL-6在突出的腰椎间盘组织明显增高,其IL-6主要由突出的腰椎间盘周围的肉芽组织所产生.     

Initial characterization of the metabolism of intervertebral disc cells encapsulated in microspheres.

Adult, canine intervertebral disc cells were isolated with a sequential digestion of pronase and bacterial collagenase. The nonchondrodystrophoid nucleus pulposus exhibits two populations of cells: large notochordal cells and smaller chondrocyte-like cells. The cells from the transition zone and anulus fibrosus are uniform in size, ranging from 17 to 21 microns. The isolated cells were encapsulated in alginate beads and cultured in Ham's F-12 medium containing 5% heat-inactivated fetal bovine serum. Alginate bead formation requires calcium ions and can be reversed with a suitable chelator, thus releasing viable cells. We observed that 58% of the newly synthesized proteoglycans formed large-molecular-weight aggregates with hyaluronic acid. The proteoglycans contained low amounts of keratan sulfate (KS) (less than 5% of the total glycosaminoglycans synthesized). The chondroitin sulfates (CS) consisted of 51-67% as 6-O-sulfate and 29-39% as 4-O-sulfate, with the remainder (4-10%) present as 4,6-sulfate for all three zones of the disc. The majority of cells synthesized significant amounts of matrix as evidenced by Alcian Blue staining. By immunohistochemical analysis, the matrix contained chondroitin 6-sulfate as demonstrated by monoclonal antibodies to the unsaturated disaccharides remaining on the proteoglycan core after chondroitinase ABC digestion. Keratan sulfate was also present in the majority of the matrices around cells. These results emphasize the similarity of the newly synthesized proteoglycans secreted by cells grown in alginate beads to those synthesized by the neonate disc. These experiments also demonstrate the usefulness of this method as a microculture technique for disc cells.     

Human nucleus pulposus cells react to IL-6: independent actions and amplification of response to IL-1 and TNF-α

STUDY DESIGN.: Human nucleus pulposus cells were activated with IL-6 plus IL-6 soluble receptor (sR) in the presence or absence of IL-1β or TNF-α. Cell production of factors modulating the anabolic/catabolic balance of the disc and proteoglycan synthesis were determined. OBJECTIVE.: To evaluate NP cell response to exogenous IL-6, and how IL-6 modulates IL-1 and TNF-α actions in these cells. SUMMARY OF BACKGROUND DATA.: Interleukin-6 (IL-6) is produced by cervical and lumbar herniated discs and is associated with neurological symptoms of intervertebral disc degeneration. It upregulates catabolic gene expression and downregulates matrix protein gene expression in chondrocytes. However, no studies have evaluated the effects of IL-6 on disc nucleus pulposus (NP) cells. METHODS.: NP cells from degenerated human discs were expanded in monolayer, maintained in alginate bead culture, and activated with IL-6 plus IL-6 soluble receptor (sR), in the presence or absence of IL-1β or TNF-α. Conditioned media was collected and analyzed for nitrite, PGE-2, TIMP-1, MMP-3, VEGF, and IL-8. Proteoglycan synthesis was assayed as S-sulfate incorporation normalized to DNA content and relative gene expression measured by rtPCR. RESULTS.: IL-6 + sR decreased collagen and aggrecan message, proteoglycan synthesis, and exacerbated the downregulation of proteoglycan synthesis effected by IL-1. PGE-2 synthesis was increased by IL-6 + sR, as was the induction of COX-2 mRNA. IL-6 + sR also enhanced IL-1 and TNF-α stimulated synthesis of PGE-2. IL-6 + sR induced MMP-3 approximately twofold and increased gene expression and synthesis in cells exposed to IL-1 and TNF-α. MMP-13 induction by TNF-α was also potentiated by IL-6 + sR. IL-6 + sR induced IL-6 gene expression and increased that stimulated by TNF-α fourfold. CONCLUSION.: The results suggest maneuvers to diminish IL-6 production in the disc could provide some protection against the adverse effects of IL-1 and TNF-α, thus, helping preserve disc composition, structure, and function.     

Osteogenic protein-1 is most effective in stimulating nucleus pulposus and annulus fibrosus cells to repair their matrix after chondroitinase ABC-induced in vitro chemonucleolysis.

BACKGROUND CONTEXT: Chondroitinase ABC (C-ABC) is used in chemonucleolysis to degrade, with great specificity, the chondroitin sulfate and dermatan sulfate chains of proteoglycans (PGs). A recent study showed that osteogenic protein-1 (OP-1) is very effective in stimulating the production and formation of the extracellular matrix by rabbit intervertebral disc cells. PURPOSE: To test the hypothesis that the repair of the extracellular matrix of the intervertebral disc after chemonucleolysis by C-ABC can be stimulated by exposure to a low dose of a growth factor, OP-1. STUDY DESIGN: An alginate bead cell culture system was used to monitor the effects of OP-1 on the repair of damaged matrices after in vitro chemonucleolysis with C-ABC. METHODS: Rabbit nucleus pulposus (NP) or annulus fibrosus (AF) cells cultured for 2 weeks in alginate gel were briefly exposed to low concentrations of C-ABC and then cultured in the presence or absence of OP-1. The control group was cultured without enzyme treatment for the same period in the absence of OP-1. At each time point, the contents of DNA and proteoglycan accumulation and proteoglycan synthesis were measured. RESULTS: NP or AF cells cultured in alginate beads, which were digested with C-ABC and then treated with OP-1, recover PG content more rapidly than those cultured in the absence of OP-1. The major contributor to the superior matrix repair in the cells treated with OP-1 was an up-regulation of proteoglycan synthesis. CONCLUSIONS: OP-1 was effective in stimulating matrix repair by NP and AF cells after their matrices were nearly totally depleted of sulfated glycosaminoglycans. The use of OP-1 after chemonucleolysis might help the disc to regain biomechanical strength, weakened by enzyme digestion, by stimulating matrix metabolism.     

腰椎间盘蛋白多糖核心蛋白的基因表达

目的　探讨在椎间盘中核心蛋白ｍＲＮＡ 表达的增龄性变化。方法　通过ＲＴ－ ＰＣＲ制备核心蛋白特异性的ｃＤＮＡ 探针,应用原位杂交的方法,分别对胎儿和成人椎间盘标本进行核心蛋白ｍ ＲＮＡ表达的定位观察。结果　在胎儿椎间盘标本中,髓核内呈现高表达,纤维环和髓核交界处表达减弱,纤维环处未见阳性表达信号。在成人标本中,髓核和纤维环均未见明显的阳性表达。结论　在胎儿椎间盘核心蛋白主要在髓核高表达,从髓核到纤维环其表达依次降低。     

Biochemical and immunological study of lumbar disc degeneration

Proteoglycans of 18 lumbar discs obtained at surgery for lumbar disc lesion were studied biochemically and immunologically (using monoclonal antibody that recognizes core protein of annulus fibrosus proteoglycans) and compared with the results of similar studies on discs obtained at autopsy and Zielke's operation for scoliosis. Disc proteoglycans showed a decrease in extraction and hexuronic acid/protein weight ratio with age in both surgical and post-mortem specimens. Although the molecular weight of proteoglycan monomer seemed to show a slight decrease, that of chondroitin sulfate chain showed no change with age in surgical discs. The proportion of proteoglycan aggregate showed a decrease with age until 40 years old, it went up thereafter because of the loss of proteoglycan monomers. This biochemical degeneration paralleled discogramic degeneration and monoclonal antibody that recognized core protein showed a strong affinity with severely degenerated discs. Besides aging, lumbar instability seemed to exert a profound influence on the progression of degeneration.     

The short-term effects of electrosurgical ablation on proinflammatory mediator production by intervertebral disc cells in tissue culture.

BACKGROUND CONTEXT: Percutaneous discectomy can be performed by a variety of methods. One method, electrosurgical ablation, has been shown in a chronic animal model to alter the expression of inflammatory cytokines in degenerated discs. PURPOSE: To determine whether electrosurgical ablation has an acute direct effect on proinflammatory mediator production by disc cells. STUDY DESIGN: A short-term in vitro study using normal and interleukin (IL)-1alpha stimulated porcine disc cells cultured in alginate gel to evaluate the biochemical effects of electrosurgical ablation. METHODS: Porcine annulus and nucleus cells were embedded into alginate gels and cultured using control culture media or IL-1alpha-treated media for 6 days before ablation treatment. Treated gels were ablated by using a radiofrequency-based electrosurgical device for 5 seconds and cultured an additional 3 or 6 days. IL-1beta, IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), prostaglandin E2 (PGE2), nitric oxide (NO), and heat shock protein-70 (Hsp70) levels in culture medium were measured. Levels were normalized to DNA and compared between ablated and shams. RESULTS: For normal annulus cells, there were no significant changes in cytokine levels between ablation and sham groups. For normal nucleus cells, ablation produced significantly greater levels of IL-8 at 3 days and 6 days, Hsp70 at 3 days but not 6 days, and NO at 6 days. PGE2 was also increased at 3 days and 6 days but not significantly. For IL-1-stimulated annulus cells, IL-6 and NO in the ablation group were decreased at 3 days relative to the control group. However, IL-6, IL-8, PGE2, and Hsp70 were significantly increased in the 6-day ablation group. For degenerated nucleus cells, IL-6, IL-8, and TNF-alpha were significantly decreased in the ablation group at both 3 days and 6 days. Ablation resulted in reduced PGE2 at 3 days but not 6 and reduced Hsp70 and NO at 6 days. CONCLUSIONS: The results show that electrosurgical ablation has an acute direct effect on proinflammatory mediator production by disc cells. The effect produced depends on disc cell phenotype, the mediator, and time. These direct biologic effects may be a mechanism of pain relief after percutaneous discectomy using electrosurgical ablation. However, the measured responses are limited to the short-term (1 week), and the existence of a prolonged effect remains to be determined.     

The influence of human intervertebral disc tissue on the metabolism of osteoblast-like cells

Extensive studies have been performed to evaluate different factors that may affect on spinal interbody fusion, but the role of intervertebral disc tissue in the fusion process remains unclear. To study the influence of intervertebral disc tissue on osteoblast metabolism, we harvested disc tissue from patients who had undergone spinal surgery. The nucleus pulposus and annulus fibrosus were separately co-cultured with osteoblast-like cells SaOS-2 by means of culture inserts or organ culture. We assayed alkaline phosphoatase activity, 3H-thymidine incorporation into the DNA, and production of collagen type I, IL-1beta, IL-6, IL-10, and TNF-alpha. Exposure of the nucleus pulposus (NP) to osteoblast-like cells revealed stimulation of alkaline phosphatase production, 3H-thymidine incorporation and collagen type I production. Exposure of the annulus fibrosus (AF) stimulated 3H-thymidine incorporation and collagen type I production, but did not affect ALP activity. IL-6 was detected after application of NP and AF. Interleukin IL-10, IL-1beta and TNF-alpha were all below detection levels after application of disc tissue. Our findings show that frozen disc tissue stimulates the metabolism of osteoblast-like cells in vitro.