退变腰椎间盘内细胞凋亡的检测与分析

目的:从基因水平探讨椎问盘退变病因病理机制.方法:日本大耳白兔60只,分实验组和对照组各30只,建立腰椎间盘退变模型,6个月后行透射电镜观察及原位细胞凋亡检测与分析.结果:椎间盘内有细胞凋亡的发生,椎间盘细胞凋亡发生率:实验组为(34.0±2.9)%,对照组为(28.0±2.8)%.退变椎间盘内细胞凋亡率高,与对照组相比,差异显著(P＜0.01).结论:退变椎间盘内细胞有较高的凋亡率,且有明显的部位差异,椎间盘退变与细胞凋亡的调控基因有关.     

体外培养人退变髓核与纤维环细胞增殖能力的研究

目的 探讨体外培养的人退变髓核与纤维环细胞的增殖能力,比较2种退变细胞体外培养中所表现的不同生物学行为,为椎间盘退变疾病的预防与治疗提供新的理论依据.方法 采集临床腰椎间盘突出症患者手术取材的椎间盘组织标本,病理学诊断评估其退变程度,酶消化法原代培养髓核与纤维环细胞,并鉴定.每例病例的髓核细胞与纤维环细胞分别体外培养至第5代,各代次细胞传代接种密度控制为1×105个.同条件培养48 h后,观察每一代细胞的形态学变化,同时应用流式细胞仪检测2种细胞的增殖能力.结果 体外培养的退变髓核与纤维环细胞生长状态良好.随着体外传代的进行,退变髓核与纤维环细胞S期细胞百分比和增殖指数(PI)均呈上升趋势,髓核细胞PI值于第3代时达到峰值,而纤维环细胞PI值于第5代时最高;且第2～4代的退变髓核细胞的增殖活性均高于退变纤维环的增殖活性(P＜0.05).结论 体外培养的人退变髓核与纤维环细胞有着不同的增殖特点,髓核与纤维环细胞对体内退变微环境有着不同的响应机制,并影响着整个椎间盘退变的进展.     

如何构建能较全面模拟人类椎间盘退变性疾病的椎间盘退变实验动物模型

背景:椎间盘退变性疾病的发病机制目前尚无定论,建立理想的椎间盘退变动物模型是研究该病发病机制或治疗的重要途径。目的:对近年来各种椎间盘退变动物模型研究的模型选择、建模方法以及优缺点做一综述。方法:计算机检索万方数据库、知网数据库、Pub Med数据库以及FMJS数据库近5年国内外关于椎间盘退变的体内及体外动物模型的文献。结果与结论:目前的文献报道显示中小型动物是现阶段较为常用的模型动物;造模方法可以分为体内和体外模型两大类,体外模型通过细胞或组织培养,从细胞及分子水平揭示椎间盘退变的机制,体内模型则使用不同种物理或化学方法对椎间盘进行干预,模拟椎间盘退变的结果,并从干预过程中反证椎间盘退变的机制。但因人类的椎间盘退变性疾病发病机制复杂,目前尚无定论,所以目前的动物模型均不能较全面的模拟人类椎间盘退变性疾病。     

椎间盘退变模型的研究进展

椎间盘退变疾病发病率越来越高,但退变的机制尚不明确.椎间盘退变模型是现今研究椎间盘退变疾病的主要方式,退变模型主要分为体内退变模型和体外退变模型两大类.两种退变模型从不同角度研究椎间盘退变的病理生理过程,为揭示退变机制及预防、治疗椎间盘退变疾病起着重要作用.本文就目前国内外关于两种不同椎间盘退变模型研究的进展作一综述.     

正常与退变椎间盘纤维环细胞相关基因的差异表达

目的: 研究人正常和退变椎间盘纤维环细胞椎间盘退变相关基因表达量的差异,探讨这些相关基因与椎间盘退变的关系,筛选明显差异基因作为退变纤维环细胞体外刺激因子,逆转椎间盘退变。方法: 实时荧光定量PCR(real-time qRT-PCR,SYBR Green)技术检测人正常和退变纤维环细胞TGF-β、IGF-1、bFGF、IL-1、TIMP-1、COL1A1、BMP-14、PDGF、aggrecan、COL2A1、SOX 9、BMP-2、iNOS、 EGF、 MMP3、BMP-4和BMP-7 mRNA表达水平。结果: 与正常纤维环细胞相比,退变纤维环细胞TGF-β、IL-1、PDGF和IGF-1 mRNA表达量降低(P<0.01,P<0.05);bFGF、TIMP-1、BMP-14和COL1A1 mRNA表达升量高(P<0.01);正常纤维环细胞aggrecan和BMP-2 mRNA高表达,退变纤维环细胞两者表达阴性;COL2A1、iNOS和MMP3在正常纤维环细胞mRNA有表达,在退变纤维环细胞表达阴性;EGF在正常纤维环细胞mRNA 表达阴性,而在退变纤维环细胞有表达。Sox-9、BMP-4和BMP-7在正常和退变纤维环细胞mRNA表达均为阴性。结论: 椎间盘纤维环退变与TGF-β、IL-1、PDGF、IGF-1、aggrecan和BMP-2 mRNA低表达,bFGF、TIMP-1、BMP-14和COL1A1 mRNA高表达相关;BMP-2、TGF-β、PDGF和IGF-1可作为退变纤维环细胞体外刺激因子,用于逆转椎间盘退变的研究;bFGF、TIMP-1和BMP-14 可能是椎间盘纤维环细胞退变的标志物。     

GATA4调控NF-κB信号通路促进椎间盘髓核细胞发生炎症和衰老

目的观察GATA4对椎间盘退变的影响,并探究其促进髓核细胞发生炎症和衰老的可能机制。方法采用免疫组化、荧光、RT-qPCR和Western blot方法检测组织标本中GATA4在不同程度退变的椎间盘中的表达水平;Lenti-GATA4慢病毒转染髓核细胞,构建稳定表达GATA4的髓核细胞;通过Western blotting和β-Gal染色阳性计数,比较GATA4诱导髓核细胞发生炎症与叔丁基过氧化物(tert-butyl hydroperoxide,TBHP)诱导髓核细胞发生炎症的效果差异;加入NF-κB通路抑制剂,研究GATA4促进髓核细胞发生炎症和衰老的机制;构建大鼠椎间盘针刺退变模型,体内验证GATA4在促进椎间盘退变的的机制。结果严重退变的椎间盘中髓核细胞表达更多的GATA4,而且体外实验证实,过表达GATA4后,髓核细胞更易发生炎症和衰老。抑制NF-κB通路后,GATA4在促进髓核细胞发生炎症和衰老的作用被明显抑制,体内实验也证明椎间盘退变后髓细胞中GATA4和炎症因子IL-6表达都明显增加。结论 GATA4可以活化NF-κB信号通路,增加促炎细胞因子IL-6和TNF-α表达,加重椎间盘退变的进展。     

椎间盘退变的机制及修复

背景:目前为止,椎间盘退变源性腰痛的发病机制研究并不十分清楚明了,对于其治疗的手段也多种多样,但效果不一。目的:综述近年国内外椎间盘退变机制及修复的研究进展。方法:由第一作者检索至2014年11月为止Pub Med数据(http://www.ncbi.nlm.nih.gov/Pub Med)及CNKI中国期刊全文数据库(http://www.cnki.net/),以"椎间盘退变因素,椎间盘源性腰痛,椎间盘退变治疗"等为检索词,共检索到78篇相关文献,排除重复研究,共30篇文献符合纳入标准。结果与结论:综合大量国内外科研人员对椎间盘退变的研究,目前比较推崇的退变因素包括形态学上的改变、炎性递质和细胞外基质的变化、生长因子的作用等,治疗上仍处于对其形态学的修复方面,其中经皮穿刺椎间盘减压、椎体融合、人工椎间盘置换、动态稳定系统等手术治疗方法对椎间盘退变引起的症状有确切的疗效。     

椎间盘退变过程中MMP/Timp基因表达变化的研究

目的:采用新西兰大白兔的纤维环损伤制作腰椎间盘退变模型,以证实和比较在人椎间盘退变中的基因变化情况.方法:损伤L4.5、L5.6纤维环,行核磁共振及计算机扫描摄影拍片证实椎间盘退变情况,同时取椎间盘行精确定量逆转录聚合酶链式反应观测MMP/Timp系列基因的变化.结果:证实了兔腰椎纤维环损伤后腰椎逐渐退变,且与人类退变结果相似,基因表达情况MMP-1、MMP-2、MMP-3、Timp-1、Timp-2早期均上调,MMP-3、Timp-1在退变的晚期出现下调.结论:人类腰椎间盘退变中明显上调的基因在此退变模型椎间盘中被发现同样上调,从而在分子水平证实了此动物退变模型与人类的相似性.     

体外培养人正常椎间盘髓核细胞与退变髓核细胞的生物学性状比较:可以为干预退变髓核细胞提供最佳时机吗?

背景:人体椎间盘是一个承受载荷却又缺乏血管的结构,因而容易发生退行性变,但椎间盘退变的机制尚不明确。目的:通过体外培养人正常椎间盘髓核细胞与退变髓核细胞生物学性状比较,认识退变髓核细胞的细胞学退变时机。方法:分离、培养人正常及退变椎间盘髓核细胞,对两种细胞采用光镜、电镜等形态学方法进行大体形态和超微结构观察,采用生长曲线和XTT实验研究两种细胞的生长动力学差异,测定髓核细胞的活力和细胞Ⅱ型胶原及糖胺多糖的mRNA表达。结果与结论:退变椎间盘细胞至少要比正常椎间盘细胞提前2代出现形态学老化表现。退变髓核细胞表现为G1期阻滞,使细胞不能进入S期,细胞有丝分裂受到抑制。退变髓核细胞总体来说,生长要比正常髓核细胞快,但老化也较快。退变髓核细胞自第1代开始,Ⅱ型胶原和糖胺多糖的mRNA表达比同期正常髓核细胞低得多。说明在体外培养条件下,退变髓核细胞持续增殖能力低,更容易衰老、凋亡。提示退变髓核细胞体外培养衰老较快,进行干预试验逆转椎间盘退变的最佳时机为传2代之前的细胞。     

尼古丁在人椎间盘髓核细胞退变中的作用

目的探讨不同浓度及时间的尼古丁在人椎间盘髓核细胞退变中的作用。方法随机选择5例腰椎骨折患者的椎间盘髓核组织进行体外细胞培养,种于12孔板中,按照0、1、33.3、100、200、500ng/m L的尼古丁浓度以及1、2、3、7、10天加入来培养,每组细胞用Trizol法提取总RNA,用荧光定量PCR测定AQP1,AQP3和II型胶原(Collagen)以及蛋白多糖(Aggrecan)的m RNA表达。用SPSS19.0进行统计分析。结果随着尼古丁浓度升高及培养时间的延长,髓核细胞形态变化明显,贴壁能力减弱,胞质减少,胞核萎缩,空泡形成,凋亡增加。且细胞AQP1及AQP3的m RNA水平随着尼古丁浓度及时间的增加而降低,II型胶原及蛋白聚糖m RNA水平也降低,细胞发生退变。结论尼古丁会降低人椎间盘髓核细胞AQP1和AQP3的表达,进而降低髓核细胞II型胶原和蛋白聚糖的表达,促进椎间盘退变,且作用时间越长,浓度越高,细胞退变越严重。     

Cyclic straining of cell-seeded synthetic ligament scaffolds: development of apparatus and methodology

Cyclic tensile strains acting along a ligament implant are known to stimulate cells that colonize it to proliferate and to synthesize an extracellular matrix (ECM), which will then remodel and form a new ligament structure. However, this process of tissue induction is poorly understood. As a first step toward elucidating this process, we aimed to investigate the effect of cyclic tensile strain on the proliferation of, and possible ECM synthesis by, cells colonizing ligament scaffolds. Because there was no commercially available apparatus to undertake such investigation the objectives of this study were to develop an apparatus for the application of cyclic tensile strains on cell-seeded synthetic ligament scaffolds and to develop and validate (through preliminary data obtained using the apparatus) methodology for studying the effect of cyclic strain on cell proliferation. We designed a multi-station test apparatus that operated inside an incubator. It allowed the application of tensile cyclic strains of between 0.5% and 5% at a frequency of 1 Hz on cell-seeded polyester ligament scaffolds immersed in culture medium. Test stations with windows in their bases could be easily de-coupled from the apparatus. This allowed monitoring of cell proliferation and morphology, with inverted light microscopy, through the transparent glass bases of the culture wells. Preliminary experiments lasting for 1 day or 9 weeks examined the effect of selected aspects of the cyclic strain on proliferation of cells seeded onto ligament scaffolds. Tests lasting for 1 day showed that the application of cyclic tensile strain of 5% for 4 h increased cell proliferation 24% above that observed in unstrained controls (p < .05). Scanning electron microscopy data from tests lasting for 9 weeks demonstrated further the dependency of cell proliferation and possible ECM synthesis on the magnitude of the strain. The larger the amplitude, the greater was the coverage of the scaffold with cells and ECM. Transmission electron microscopy of the ECM observed at 9 weeks showed evidence of collagen fibrils aligned in the direction of load in strained scaffolds, whereas the tissue on the control scaffolds was random.     

ERK信号通路参与调控周期性张应变诱导的人牙周膜细胞增殖

目的 探讨周期性张应变对人牙周膜细胞 （human periodontal ligament cell , hPDLCs ) 增殖的影响及其相关机制。方法 应用FX-4000T 细胞应变加载系统,对体外培养的人牙周膜细胞施加周期性张应变,幅度分别为10 % 和20 %,加载时间为6 h和24 h,频率均为0.1 Hz,以未加载的静态细胞作为对照组。Western blot 法检测细胞增殖细胞核抗原（PCNA）和细胞内信号转导分子p-ERK1/2表达的变化。用ERK1/2的特异性抑制剂PD 98059预处理细胞后,在0.1 Hz,10 % 幅度条件下,加载6 h,检测p-ERK1/2的表达水平变化对细胞PCNA表达的影响。 结果 与静态组相比,周期性张应变诱导人牙周膜细胞的PCNA和p-ERK1/2表达增加,10 % 幅度和20 % 幅度相比无显著性差异。同一幅度张应变,加载6 h和24 h对细胞PCNA和p-ERK1/2表达的影响无显著性差异。ERK1/2的抑制剂PD98059不仅可以明显抑制张应变诱导的p-ERK1/2 活化,而且张应变诱导的细胞PCNA表达也被明显抑制。结论 周期性张应变可激活ERK信号通路,促进体外培养人牙周膜细胞的增殖。     

An annulus fibrosus closure device based on a biodegradable shape-memory polymer network

Injuries to the intervertebral disc caused by degeneration or trauma often lead to tearing of the annulus fibrosus (AF) and extrusion of the nucleus pulposus (NP). This can compress nerves and cause lower back pain. In this study, the characteristics of poly(d,l-lactide-co-trimethylene carbonate) networks with shape-memory properties have been evaluated in order to prepare biodegradable AF closure devices that can be implanted minimally invasively. Four different macromers with (d,l-lactide) to trimethylene carbonate (DLLA:TMC) molar ratios of 80:20, 70:30, 60:40 and 40:60 with terminal methacrylate groups and molecular weights of approximately 30 kg mol⁻¹ were used to prepare the networks by photo-crosslinking. The mechanical properties of the samples and their shape-memory properties were determined at temperatures of 0 °C and 40 °C by tensile tests- and cyclic, thermo-mechanical measurements. At 40 °C all networks showed rubber-like behavior and were flexible with elastic modulus values of 1.7–2.5 MPa, which is in the range of the modulus values of human annulus fibrosus tissue. The shape-memory characteristics of the networks were excellent with values of the shape-fixity and the shape-recovery ratio higher than 98 and 95%, respectively. The switching temperatures were between 10 and 39 °C. In vitro culture and qualitative immunocytochemistry of human annulus fibrosus cells on shape-memory films with DLLA:TMC molar ratios of 60:40 showed very good ability of the networks to support the adhesion and growth of human AF cells. When the polymer network films were coated by adsorption of fibronectin, cell attachment, cell spreading, and extracellular matrix production was further improved. Annulus fibrosus closure devices were prepared from these AF cell-compatible materials by photo-polymerizing the reactive precursors in a mold. Insertion of the multifunctional implant in the disc of a cadaveric canine spine showed that these shape-memory devices could be implanted through a small slit and to some extent deploy self-sufficiently within the disc cavity.     

周期性张应变对破骨前体细胞和破骨细胞增殖和抗酒石酸酸性磷酸酶的影响

目的研究不同强度的力学载荷对破骨细胞及其前体细胞增殖、分化和功能的影响。方法以破骨诱导液培养RAW264.7破骨前体细胞,同时施加3 d的周期性张应变,然后培养4 d;另外一组RAW264.7细胞以破骨诱导液培养4 d,将其诱导为破骨细胞,再施加3 d的周期性张应变。结果在不同张应变下,两组细胞增殖活性的变化大致相同,但细胞抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatage,TRAP)活性和破骨细胞(TRAP阳性多核细胞)数量的变化明显不同。在2 500με的中等强度张应变下,第1组的TRAP活性降幅和破骨细胞数量减幅均最高,而后者TRAP活性降幅和破骨细胞数量减幅均最低。结论不同张应变对分化初期破骨前体细胞和已分化出破骨细胞的破骨前体细胞的破骨分化和功能状态的影响有明显差异。     

张力刺激对关节软骨细胞β-肌动蛋白mRNA表达的影响

目的 观察张力刺激对关节软骨细胞β-肌动蛋白(β-aetin)mRNA表达的影响.方法 分别获取正常和骨关节炎关节软骨细胞,给予3%、6%和15%的张力刺激,运用实时定量PCR方法检测不同张力刺激强度下关节软骨细胞β-actin mRNA表达的变化.结果 6%和15%的张力刺激均可明显提高lE常关节软骨细胞β-actinmRNA表达(1.34±0.05,1.36±0.04,P＜0.05),而只有15%的张力刺激才能明显增加骨关节炎关节软骨细胞β-actin mRNA表达.结论 关节软骨细胞β-actin mRNA表达量随张力刺激强度不同而变化,不同内外环境使关节软骨细胞对张力刺激的反应不同,故在其相关生物力学研究中不能应用β-actin作为内参.     

Effect of cyclic mechanical strain on glycosaminoglycan and proteoglycan synthesis by heart valve cells

Heart valves are presumed to remodel their extracellular matrix upon application of mechanical strains. In this study, we investigated the effect of cyclic tensile strain on valvular interstitial cells’ synthesis of glycosaminoglycans (GAGs) and proteoglycans (PGs), which are altered during myxomatous degeneration. Interstitial cells were isolated from mitral valve leaflets and chordate, and seeded separately within three-dimensional collagen gels. Cell-seeded collagen gels were then subjected to cyclic strains of 2%, 5% or 10% at 1.16 Hz for 48 h using a custom-built stretching device. The application of cyclic strains reduced the total GAGs retained within collagen gels in a magnitude-dependent manner for both leaflet and chordal cells. With increasing strain magnitude, however, secretion of total GAGs into the medium was reduced for leaflet cells and elevated for chordal cells. Retention of 4-sulfated GAGs increased with increasing strain magnitude for both cell types; for the chordal samples, retention of 6-sulfated GAGs was reduced at higher strain magnitudes. Compared to statically constrained or unconstrained conditions, the application of cyclic strain reduced the secretion of 6-sulfated GAGs by both cell types, and elevated secretion of 4-sulfated GAGs by leaflet cells only. Retention of the PG biglycan and secretion of the PG decorin was significantly reduced at 10% strain compared to 2% strain. In addition, there were numerous differences in the strain-dependent retention and secretion of GAGs and PGS within the leaflet and chordal groups. These results demonstrate that GAG and PG synthesis by VICs is regulated by cyclic stretching conditions.     

力学应变对大鼠骨髓间充质干细胞增殖和成骨分化能力的影响

探讨周期性双轴力学应变对大鼠骨髓间充质干细胞(M esenchym a l stem ce lls,M SC s)增殖和成骨分化能力的影响。选用9月龄健康SD雌性大鼠,分离股骨、胫骨提取骨髓,采用密度梯度离心法分离M SC s。体外培养M SC s传至第3代,以1×105细胞浓度接种于双轴力学应变系统,选取4 000μstra in,频率为1hz的力学应变对M SC s加载。每天加载3次,每次2 h,间隔2 h。观察力学应变作用后1 d、3 d,M SC s增殖和成骨分化能力的变化,并与相应未加力学应变对照组比较。结果表明:(1)力学应变可增高M SC s的碱性磷酸酶(ALP)和骨桥蛋白(OPN)表达量;力学应变作用3 d后M SC s的ALP和OPN表达量明显高于力学应变作用1 d。I型胶原(COL I)仅在力学应变作用3 d增高;骨钙素(OCN)在各组无明显变化。(2)力学应变可促进M SC s增殖,但力学应变作用1 d和3 d对M SC s增殖的作用无明显差异。上述结果提示:力学应变可以促进M SC s的增殖和成骨分化能力。     

脊神经前根中神经细胞突起的去向

分别切断15只成年猫的前、后根及颈、腰部交感干,观察到前根细胞的溃变,初步查明了前根细胞突起的去向。 1.断前根组18条前根中发现31个细胞,其中有18个溃变细胞,占58％;断后根组11条前根中发现116个细胞,其中有80个正常细胞,占69％。这说明大多数前根细胞的中枢突是经前根进入脊髓的。 2.断前根组的31个细胞中,有5个正常细胞,占16％,断后根组的116个细胞中有15个溃变细胞,占12.9％。这一事实说明前根细胞的中枢突,也有经后根入脊髓的。 3.断腰交感干组的14条前根中发现132个细胞,其中溃变细胞为77个,占58％,断颈交感干组的9条前根中发现63个细胞,其中37个为溃变细胞,占60％。由此证明半数以上的前根细胞的周围突是通过交感神经分布的。 4.本文就前根细胞突起可能存在的其他去向也进行了讨论。     

Dependency of disc degeneration on shear and tensile strains between annular fiber layers for complex loads

BackgroundOne of the first signs of disc degeneration is the formation of circumferential tears within the annulus fibrosus. It is assumed that high shear and tensile strains between the lamellae mainly cause the initiation of these failures. However, it is not known which load application and which degree of disc degeneration could lead to the highest strains and therefore, might induce the formation of tears. Therefore, the aim of this finite element (FE) study was, to find load combinations that would yield highest shear and tensile strains in differently degenerated discs.Materials and methodsA three-dimensional FE-model of a motion segment L4-5 was utilized in different degrees of disc degeneration (healthy, mild, moderate, and severe). The degenerated models consider the reduction of disc height, endplate curvatures, the osteophyte formation, the increase of nucleus compressibility, and the decrease of fiber and ligament stiffness. An axial compression load of 500 N together with moments of 7.5 Nm in single and combined load directions were simulated.ResultsHigh strains for the healthy and degenerated discs were predicted for load combinations, particularly for the combination of lateral bending plus flexion or extension. The maximum strains were located in the postero-lateral region of the disc. In comparison to the healthy disc, the maximum strains increased slightly for the mildly and moderately degenerated disc. Strains decreased strongly for the severely degenerated disc. With progressive degeneration, the size of the region of maximum strains diminished and the location transferred from the inner annulus to the adjacent bony endplates.ConclusionsThe results could be a possible explanation for the initiation of circumferential tears. The mildly degenerated disc model, which represents early stages of life, suggests that circumferential tears could primarily occur at these stages, especially for the load combinations of lateral bending plus axial rotation and lateral bending plus flexion.