Phototherapy suppresses inflammation in human nucleus pulposus cells for intervertebral disc degeneration

The etiology of intervertebral disc (IVD) degeneration accompanied by low back pain (LBP) is largely unknown, and there are no curative therapies. Painful IVD degeneration is associated with infiltrated macrophage-mediated inflammatory response of human nucleus pulposus (NP) cells. The present study aimed to address the hypothesis that pro-inflammatory cytokines derived from macrophages lead to the altered molecular phenotype of human NP cells and to investigate the effects of phototherapy (630, 525, 465 nm with 16, 32, 64 J/cm²) on pain-related cytokine interleukin (IL)-6 and chemokine IL-8 under inflammatory conditions in human NP cells. Human NP cells were treated with soluble factors derived from macrophages in an inflammatory microenvironment, similar to that found in degenerative IVD. Human NP cells were also treated with phototherapy (630, 525, 465 nm with 16, 32, 64 J/cm²), and their cytokine and chemokine levels were detected. The soluble factors caused modulated expression of IL-6, IL-8, and matrix metalloproteinases (MMPs) at the gene and protein levels, causing a shift toward matrix catabolism through the expression of MMPs and increased pain-related factors via preferential activation of the nuclear factor-kappa B (NF-κB) p50 protein. Importantly, phototherapy attenuated the protein and gene expression of pain-related factor IL-6 at all doses and wavelengths. Interestingly, phototherapy also modulated the protein and gene expression of IL-8, which is responsible for the anabolic response, at a wavelength of 465 nm at all doses, in human NP cells. These findings suggested that phototherapy, at an optimal dose and wavelength, might be a useful therapeutic tool to treat IVD degeneration.     

Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration: an in vitro and in vivo experimental study.

Reinsertion of autogenous nucleus pulposus, an innovative method to delay further disc degeneration, has been proved with an experimental animal model. This study examined whether coculture of nucleus pulposus cells with annulus fibrosus cells (a) activates annulus fibrosus cells and (b) retards disc degeneration when reinserted into the disc in a rabbit model of disc degeneration. Coculture of the two cell types stimulated proliferation of each, as indicated by increased DNA synthesis measured by increases in DNA polymerase alpha expression and uptake of 5-bromo-2'deoxy-uridine assessed by an enzyme-linked immunosorbent assay. In a model of disc degeneration in rabbits, reinsertion of activated nucleus pulposus cells delayed the formation of clusters of chondrocyte-like cells, the destruction of disc architecture, and the elaboration of type-II collagen as measured immunohistochemically compared with no treatment. The direct reinsertion of activated nucleus pulposus cells into the disc offers a promising line of investigation for delaying intervertebral disc degeneration, although these results obtained with notochordal cells may not necessarily apply when mature central nucleus pulposus cells are used.     

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.     

Fas ligand expression on human nucleus pulposus cells decreases with disc degeneration processes

Background The intervertebral disc has been reported to be an immunologically privileged environment, possibly mediated by Fas ligand (FasL) expression. On the other hand, recent studies have shown the infiltration of host immune cells into the degenerated disc, which may indicate the failure of the immune-privilege feature of the disc with degeneration. However, the relationship between FasL expression and disc degeneration is still unclear. Therefore, the purpose of this study was to clarify the relationship between FasL expression and disc degeneration. Methods Ten human degenerated disc specimens were obtained from spondylolisthesis patients and ten nondegenerated discs from idiopathic scoliosis patients during surgical procedures. Immunohistochemical staining was performed to determine the presence of FasL in cross-sections of those discs. Parts of the disc tissues were used to examine FasL expression quantitatively with Western blot analysis. To examine whether the change in FasL expression was influenced by aging, an animal study comparing the discs from young and old rats were performed using magnetic resonance imaging (MRI) and real-time polymerase chain reaction (PCR) assessment. Results Nucleus pulposus cells showed strong positive staining for FasL in all specimens examined. Quantitative examination demonstrated a significant decrease in FasL expression in the degenerated group compared with the nondegenerated group (average 67.6%, P<0.05). MRI showed no significant differences in the grade of disc degeneration between young and old rats, and also no significant difference in FasL mRNA in real-time PCR assay. Conclusions The current results indicate that FasL and its potential mechanism of immunological privilege could influence the protection of the intervertebral disc against degeneration.     

人退变髓核细胞诱导脂肪间充质干细胞向类髓核细胞分化的实验研究

[目的]探讨人脂肪间充质干细胞(adipose-derived stem cells,ADSCs)向类髓核细胞(nucleus pulposuslike cells,NPCs)诱导分化的可能性,为椎间盘退变的防治研究提供种子细胞。[方法]收集腰椎间盘退变患者手术中切除的脂肪组织及髓核组织,采用胶原酶消化法提取原代ADSCs和NPCs,倒置显微镜下观察共培养后ADSCs形态学变化。MTT法检测细胞增殖情况并绘制细胞生长曲线,采用孔径为0.4μm的Transwell小室建立细胞共培养体系,NPCs置于上层,ADSCs接种于下层,建立2个细胞培养组:ADSCs单独培养组和ADSCs/NPCs共培养组,甲苯胺蓝染色以检测蛋白聚糖表达情况,RT-PCR法检测细胞COL2A1、ACAN和SOX9基因表达水平。[结果]采用胶原酶消化法可较好的分离培养出人脂肪间充质干细胞和髓核细胞。与NPCs共培养后的ADSCs可被甲苯胺蓝染为天蓝色,且Ⅱ型胶原及SOX9基因表达水平上调(P0.05)。[结论]采用胶原酶消化法分离培养的ADSCs活力较好、增殖旺盛,与退变NPCs共培养后,软骨特异性标记物的表达明显增加,细胞表现出向NPCs分化的趋势,可作为椎间盘组织工程研究的种子细胞。     

腰椎间盘髓核退变的MRI表现与病理学的相关性研究

目的探讨腰椎间盘髓核退变的MRI表现与病理学之间的相关性,为临床应用MRI相对信号强度评价腰椎间盘髓核退变提供病理学依据。方法病例组标本取自腰椎间盘疾患手术患者,共91例101个椎间盘;对照组标本取自既往无明显腰痛史的年轻腰椎骨折患者和新鲜脑死亡患者,共6例16个椎间盘。所有标本均于术前测量MRI相对信号强度,并予以HE染色、Alcianblue临界电解质浓度(CEC)染色和水分含量测定,分析MRI相对信号强度与硫酸软骨素指数、MRI相对信号强度与水分含量、硫酸软骨素指数与水分含量之间的相关性。结果两组间的MRI相对信号强度、硫酸软骨素指数和水分含量差异均有显著性,而硫酸角质素指数差异无显著性。MRI相对信号强度与硫酸软骨素指数、MRI相对信号强度与水分含量、硫酸软骨素指数与水分含量均有明显相关性,其r值分别为0.659、0.752和0.592,P值均<0.001。根据髓核的水分含量和病理学改变,可将髓核退变大致分为四个阶段,每个阶段的MRI相对信号强度依次为:>0.82,0.73~0.82,0.64~0.72,<0.64。结论MRI相对信号强度不仅能反映髓核的水分含量,而且还能反映髓核退变过程中的病理特征。MRI相对信号强度四级定量分级法是一种评价髓核退变较好的方法。     

Alpha-smooth muscle actin expression in intact and ruptured human anterior cruciate ligament--an immunohistochemical assessment.

The objective of this work was to evaluate the distribution of alpha-smooth muscle actin cells in the intact human anterior cruciate ligament and to determine if rupture of the anterior cruciate ligament and the time since injury has any influence on the occurrence of cells with contractile capability, positively stained for a-smooth muscle actin. The intact anterior cruciate ligament group (group A) undergoing total knee arthroplasty, consisted of 9 patients (8 females) with mean age of 65.3 years. The anterior cruciate ligament rupture group (group B) consisted of 20 patients (18 males, 2 females) with mean age of 27.8 years. Healing time of the torn ligament in vivo lasted from 1 to 40 months and the patients were divided into 3 groups (Bi-Biii) embracing diverse time periods. All harvested anterior cruciate ligaments were sectioned in thirds so that there was a proximal, middle and distal third for each ligament. Distribution of alpha-smooth muscle actin was detected with mouse monoclonal anti-human smooth muscle actin antibody. The alpha-SMA cells density was significantly affected by the time after injury as well as the location in the ligament remnant. The decrease of alpha-smooth muscle actin cells in the proximal and middle thirds of the ligament after rupture may suggest that alpha-SMA cells are not mainly responsible for primary retraction of the ruptured human anterior cruciate ligament.     

Mechanical stress-induced apoptosis of nucleus pulposus cells: an in vitro and in vivo rat model

Background

Un-physiological loads play an important role in the degenerative process of inter-vertebral discs (IVD). In this study, we used an in vitro and in vivo rat model to investigate the mechanism of nucleus pulposus (NP) cells apoptosis induced by mechanical stress.

Methods

Static compressive load to IVDs of rat tails was used as the in vivo model. For the in vitro model, NP cells were tested under the physiological and un-physiological loading. For histological examination, apoptotic index study, and apoptotic gene expression, we also selected cytokines [bone morphogenetic protein (BMP)-2/7, insulin-like growth factor (IGF)-1, platelet-derived growth factor (PDGF)] to be analyzed.

Results

Under mechanical loading, cellular density was significantly decreased, but there was an increase of TUNEL positive cells and apoptosis index. In a dose-dependent manner; the necrosis became apparent in the un-physiologic strain. The selected cytokines (BMP-2/7, IGF-1, PDGF) can significantly reduce the percentage of apoptotic and necrotic cells.

Conclusions

We conclude that the intrinsic (mitochondrial) apoptotic pathway plays an important role in the compressive load-induced apoptosis of NP cells. Combination therapy reducing the mechanical load and selected cytokines (BMP-2/7, IGF-1 and PDGF) may have considerable promise in the treatment of spine disc degeneration.     

微RNA-210抑制人退行性变髓核细胞自噬影响椎间盘退行性变

目的探讨微RNA-210(miRNA-210)对退行性变髓核细胞自噬的影响及其参与椎间盘退行性变(IDD)进程的可能机制。方法收集24例IDD患者(IDD组)和6例腰椎骨折患者(对照组)椎间盘组织进行分离,培养髓核细胞。通过实时荧光定量聚合酶链反应(FQ-PCR)检测髓核细胞中miRNA-210的表达,通过单丹磺酰尸胺(MDC)染色和蛋白质印迹法检测细胞自噬水平。通过FQ-PCR或蛋白质印迹法检测过表达或抑制miRNA-210对细胞自噬和细胞外基质代谢的影响。通过生物信息学手段寻找miRNA-210与自噬相关的靶基因及miRNA-210的结合位点,并应用双荧光素酶报告实验验证。结果与对照组相比,IDD组髓核细胞中miRNA-210表达量增加,细胞自噬水平降低。过表达miRNA-210会抑制髓核细胞自噬,同时降低Ⅱ型胶原(ColⅡ)及蛋白多糖表达量,升高基质金属蛋白酶-3(MMP-3)和MMP-13表达量。自噬抑制剂3-MA减弱miRNA-210对ColⅡ、蛋白多糖、MMP-3和MMP-13的调节作用。miRNA-210与自噬相关蛋白7(ATG7)存在结合位点,过表达miRNA-210通过沉默ATG7抑制细胞自噬,激活MMP-3和MMP-13,促进细胞外基质(ColⅡ和蛋白多糖)降解。结论在发生退行性变的椎间盘组织中miRNA-210表达量增加。过表达的miRNA-210可能通过直接作用于靶基因ATG7抑制细胞自噬,促进细胞外基质降解,推动IDD进程。     

pH值对人退变椎间盘髓核细胞凋亡的影响

[目的]观察不同pH值对体外培养人退变椎间盘髓核细胞凋亡的影响,为椎间盘退变的预防与治疗提供理论依据。[方法]对体外培养的人退变椎间盘髓核细胞利用甲苯胺蓝和番红O染色进行鉴定,并对P3代细胞分别用普通培养基(pH值7.4)、酸性培养基(pH值6.8)和酸性培养基(pH值6.8)+Cucl_2(酸质子受体抑制剂)培养24 h,用流式细胞仪测定细胞凋亡,计算细胞凋亡率。[结果]细胞染色结果均为阳性,证明实验中所培养的细胞为髓核类软骨细胞。普通培养基组的凋亡率为(8.86±0.20)%,酸性培养基组的凋亡率为(11.23±0.77)%,酸性培养基+Cucl_2组的凋亡率为(9.60±0.49)%。与其他两组相比,酸性培养基组的凋亡率明显增加(P0.05)。[结论]在低pH值的环境下,人退变椎间盘髓核细胞的凋亡率增加。     

In vitro interaction between muscle-derived stem cells and nucleus pulposus cells

Background contextCurrent therapies for intervertebral disc degeneration (IDD) are aimed at treating the clinical symptoms arising from IDD rather than directly treating the underlying problem. Pathophysiology of IDD is characterized by a progressive decrease in proteoglycan content and cell density in the nucleus pulposus (NP). A cell-based therapy is a promising concept that uses various cell types to repopulate the disc in an attempt to slow, stop, or reverse the progressive loss of proteoglycans. Stem cells appear to be an excellent candidate for this purpose, based on their ability to differentiate into various connective tissue lineages. The muscle tissue could serve as a good source of adult stem cells because of its vast abundance through out the human body.PurposeTo examine the interaction between the nucleus pulposus cells (NPCs) and the muscle-derived stem cells (MDSCs) in vitro.Study designNPCs and MDSCs were cocultured and proteoglycan production and cell proliferation were evaluated.MethodsVarious ratios of human NPCs were cocultured for 2 weeks with murine MDSCs (transduced with retro/LacZ) in a monolayer culture. Each well contained an admixture of cells with NPC-to-MDSC ratios of 0:100, 25:75, 50:50, 75:25, 100:0. Glycosaminoglycan (GAG) content (1,9 dimethylmethylene blue [DMMB]), newly synthesized proteoglycan (³⁵S incorporation), and DNA content were measured, and cultures were stained with 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-Gal) for cell counting.ResultsThe NPC-to-MDSC ratio of 75:25 resulted in a significant increase in GAG content compared with NPCs alone. All coculture ratios showed increase in GAG content in comparison with MDSC culture alone. In addition, cocultures showed a significant increase in ³⁵S incorporation normalized to DNA content in comparison with MDSC alone.ConclusionsThe data from this study shows a synergistic effect between MDSCs and NPCs resulting in an upregulated proteoglycan synthesis and NPCs proliferation in vitro.     

腰椎间盘髓核组织中TrkB、NDRG2表达及其与衰老髓核细胞的关系

目的 探究退变腰椎间盘髓核组织中酪氨酸激酶受体B(tyrosine kinase receptor B,TrkB)、N-Myc下游调节基因2(N-myc downstream regulatory gene 2,NDRG2)表达变化,并分析两者与衰老髓核细胞之间的相关性.方法 选取2018年1月～2020年4月于本院行...     

正常髓核细胞的黏弹性研究

目的 观察正常髓核细胞的黏弹性。方法 髓核组织取材于3例脊柱侧凸矫形手术者术中取出废弃的髓核组织,用胰蛋白酶和Ⅱ型胶原酶消化分离细胞,Ⅱ型胶原免疫荧光组化和蕃红染色进行细胞鉴定,测量细胞直径,采用微管吸吮技术分析髓核细胞的黏弹性特性。结果 髓核细胞直径为(15.40±1.83)μm,正常髓核细胞的黏弹性参数k1(0.101 ±0.052) kPn、k2(0.353±0.199) kPa和μ(3.034±1.843) kPa·s。直线相关性分析表明,仅k1与髓核细胞直径明显相关(r=-0.389,P＜0.05)。结论 正常髓核细胞表现为典型的黏弹性固体蠕变特征;微管吸吮技术可以作为测量髓核细胞生物力学特性的可靠方法。     

椎间盘髓核细胞中Sox9与Ⅱ型胶原基因表达的关系

目的：探讨椎间盘髓核组织中Sox9基阏表达的变化及其与Ⅱ型胶原基因表达的关系。方法：将30个椎间盘组织按Thompson分期分为Ⅰ～Ⅳ期．应用RT—PCR、Westernblot和免疫组化方法检测各期间盘组织巾Sox9、Ⅱ型胶原基因的mRNA和蛋白表达：应用SPSS10，0统计学软件．采用单闪素方差分析、t检验和Pearson相关性检验分析两者的相互关系。结果：椎间盘髓核组织巾Sox9mRNA的表达量在总体上低于Ⅱ型胶原，Sox9蛋白表达位于细胞核中而Ⅱ型胶原主要位于细胞间质内，Sox9在ThompsonⅠ期椎间盘的细胞核内表达很强而存Ⅳ期则很弱甚至缺失：从ThompsonⅠ-Ⅳ期两种基因的mRNA和蛋白表达水平均逐渐降低，各分期间有显著性差异（P〈0．05）。ThompsonⅠ～Ⅳ期标本巾Ⅱ型胶原与Sox9表达量的下降趋势相近。结论：Sox9和Ⅱ型胶原基因表达水平与Thompson分期密切相荚，随椎间盘退变程度加重表达逐渐降低，且两者下降趋势相近。     

腰椎间盘突出症有限手术治疗及髓核病理观察相关分析

自2002年1月至2006年3月，采用有限手术治疗腰椎间盘突出症297例，对手术摘除的髓核标本进行病理检查，探讨突出髓核组织与临床发病及相关情况。     

人髓核间充质干细胞的分离提纯方式及生物学活性鉴定

目的 :探索人髓核间充质干细胞(nucleus pulposus mesenchymal stem cells,NPMSCs)的提纯方法并鉴定其生物学活性。方法:收集3例腰椎间盘突出症患者的退变髓核组织(Pfirrmann分级均为Ⅳ级),利用酶消化法分离细胞。采用两种方法分离提纯NPMSCs,一组细胞采用贴壁法培养(贴壁组),另一组通过流式细胞分选技术利用NPMSCs表面阳性标志物CD73、CD90、CD105获得NPMSCs(流式组)。将两种方法获得的NPMSCs进行体外培养扩增,分别进行形态学观察,细胞计数试剂盒(Cell Counting Kit,CCK-8)检测增殖能力。贴壁组NPMSCs采用流式细胞分选仪在进行分选之前检测免疫表型,流式组NPMSCs在生长达80%~90%融合时进行免疫表型的检测。向成骨、成脂、成软骨诱导分化,诱导28d后分别进行茜素红染色观察其成骨能力、油红O染色观察其成脂能力、甲苯胺蓝染色观察其成软骨能力,利用Imag J软件计算染色区域所占的面积百分比。比较两组NPMSCs在形态学、免疫表型及增殖和分化能力的差异。结果:形态学观察发现,两组NPMSCs均呈漩涡状生长,贴壁组NPMSCs可见散在的单个细胞生长;流式组NPMSCs长梭形形态更长,排列更加紧密,少见散在的单个贴壁生长细胞。流式细胞分选后所得的NPMSCs占细胞总数的(89.67±2.52)%,可以进行体外培养扩增,细胞为典型的长梭形特征,漩涡状生长,在接种后12~15d达80%~90%融合,增殖能力在接种后5~13d明显高于贴壁组NPMSCs(P0.05)。流式组NPMSCs的CD73、CD90、CD105的表达率明显高于贴壁组NPMSCs(P0.05),并且低表达CD34、CD45及HLA-DR。两种方法获得的NPMSCs均能完成三系诱导分化,流式组成骨、成脂、成软骨染色区域百分比均明显高于贴壁组(P0.05)。结论:利用流式细胞分选技术从人退变髓核组织中可获得较高纯度的NPMSCs,并能进行后续培养扩增。与贴壁法获得的NPMSCs相比,流式细胞分选的NPMSCs具有更强的增殖与分化能力。