慢病毒介导的RNA干扰技术构建人胚椎间盘髓核上皮膜蛋白-1基因低表达细胞模型

目的:构建人胚椎间盘髓核上皮膜蛋白-1基因低表达细胞模型,为进一步研究EMP-1在椎间盘退变中的作用机制提供理想的研究平台。方法:利用慢病毒载体介导的小发夹状RNA(small hairpin RNA,shRNA)转入人胚胎肾细胞(293FT)包装慢病毒,48h后在荧光显微镜下观察绿色荧光蛋白表达情况,72h后收集病毒上清并感染人胚椎间盘髓核细胞,Western blot、RT-PCR鉴定基因干扰效果,凝胶成像分析软件对条带显色程度进行分析,得到EMP-1/GAPDH的平均值。结果:重组慢病毒滴度测定约为1×107U/ml,感染靶细胞后得到EMP-1基因低表达细胞模型,EMP-1mRNA表达水平比正常细胞下降了50%。半定量RT-PCR结果显示EMP1/GAPDH的平均值由0.46±0.03降至0.32±0.01,Westernblot结果显示EMP1/GAPDH的平均值由0.50±0.01降至0.25±0.01。结论:通过慢病毒包装技术,成功构建人胚椎间盘髓核上皮膜蛋白-1基因低表达细胞模型。     

1-磷酸鞘氨醇受体在不同退变程度髓核组织中的表达变化

目的 :比较不同退变程度人椎间盘髓核组织中3种1-磷酸鞘氨醇受体(S1PR1/2/3)表达水平的差异,探讨椎间盘中S1PR表达水平与椎间盘退变的关系。方法:收集腰椎间盘退行性病变患者手术切除的椎间盘组织,其中轻度退变(Pfirrmann分级Ⅳ级)22例,严重退变(Pfirrmann分级Ⅴ级)14例;同时取6例无椎间盘退变患者(单纯腰椎椎体骨折,Pfirrmann分级Ⅱ级)手术切除的椎间盘组织作为对照组;通过HE染色以及Saf-O染色观察不同退变程度椎间盘的组织学变化,免疫组化检测不同退变程度组织中的S1PR表达水平;Ⅱ型胶原酶消化分离提取原代髓核细胞,通过Real-time PCR、Western-bolt检测不同退变程度椎间盘髓核细胞中S1PR的表达水平,并通过细胞免疫化学方法对S1PR进行定位。结果:HE染色及Saf-O染色结果显示退变椎间盘的纤维环出现破损,髓核细胞形成明显的集落,细胞外基质减少。免疫组化结果显示正常和轻度退变的髓核组织中3种受体(S1PR1/2/3)都有表达,严重退变的组织中表达极弱;Real-time PCR结果显示对照组髓核细胞中S1PR1/2/3的m RNA表达水平分别是严重退变组的5.34±0.52倍、7.25±0.04倍、1.92±0.06倍,轻度退变组S1PR1/2/3的m RNA表达水平分别是严重退变组的4.35±2.45倍、4.96±3.44倍、2.19±0.82倍;Western-blot发现对照组和轻度退变组髓核细胞中S1PR1/2/3均有表达,严重退变组表达水平较低;免疫细胞化学显示S1PR主要集中在髓核细胞的细胞质和细胞膜上。结论:髓核组织中主要表达S1PR1/2/3,在严重退变的髓核组织和细胞中其表达水平明显下降,S1P及其受体可能参与椎间盘髓核组织的退变过程。     

人胚椎间盘细胞体外培养模型的建立和鉴定

目的通过建立人胚椎间盘细胞体外培养模型,进一步研究细胞因素在椎间盘退变中的作用机制。方法取4例人体胚胎腰椎间盘,在改良Eagle培养基中建立椎间盘细胞模型,细胞染色、逆转录聚合酶链式反应、免疫荧光检测细胞Ⅰ、Ⅱ型胶原的表达进行鉴定。结果椎间盘细胞可在体外培养并进行传代,细胞中Ⅰ、Ⅱ型胶原具有较高的表达,细胞具有较高的分化状态。结论源自胚胎的髓核细胞于体外环境中可以较长时间维持其表型不变,且表型与正常髓核细胞很接近。     

成人退变性椎间盘髓核细胞体外培养及形态学观察

目的通过对成人退变椎间盘髓核细胞的体外培养和形态学观察,进一步研究细胞因素在椎间盘退变中的作用机制。方法取5例患椎间盘突出症并行椎间盘摘除手术的成人髓核,分离后在培养基中进行髓核细胞培养,细胞染色、逆转录聚合酶链式反应、免疫荧光检测细胞Ⅰ、Ⅱ型胶原的表达。结果椎间盘髓核细胞可在体外培养,30d后方可进行传代,最佳的培养条件为胎牛血清/培养基体积分数为10%～20%,pH值7.0。髓核细胞中出现Ⅰ型胶原,并具有较高的表达,Ⅱ型胶原表达微弱。结论成人退变髓核细胞体外培养时间较长,细胞增殖能力低下,特定培养条件的摸索是成功与否的关键。     

单纯Ⅱ型胶原酶消化法分离、培养人退变椎间盘髓核细胞的形态学观察

目的:探讨单纯Ⅱ型胶原酶消化法体外培养扩增人退变椎间盘髓核细胞的可行性。方法:收集20例人退变椎间盘髓核,单纯Ⅱ型胶原酶消化分离出髓核细胞并连续培养传代,倒置相差显微镜和HE染色观察细胞形态学变化,甲苯胺蓝染色检测髓核细胞内聚集蛋白聚糖的表达,免疫细胞化学法行Ⅱ型胶原染色,观察髓核细胞的类软骨表型表达情况。结果:单纯Ⅱ型胶原酶消化法可较好的分离培养人退变椎间盘髓核细胞,20例人退变椎间盘髓核,培养成功16例;原代髓核细胞平均7d贴壁,呈类圆形或多角形,P1代髓核细胞平均12h贴壁,呈大梭形或多角形,两代细胞融合95%所需时间分别为30d和7d,差异有统计学意义(P0.01);聚集蛋白聚糖和Ⅱ型胶原主要表达于原代和P1代髓核细胞浆内,被甲苯胺蓝染成天蓝色,免疫细胞化学染色主要表现为黄褐色沉淀,两代间聚集蛋白聚糖和Ⅱ型胶原的表达无统计学差异(P0.05)。结论:单纯Ⅱ型胶原酶消化法可简化髓核细胞分离步骤,提高培养效率;传一代后髓核细胞增殖速率提高,但仍维持类软骨表型,表达聚集蛋白聚糖和Ⅱ型胶原。     

白藜芦醇与椎间盘退变性疾病

椎间盘退变性疾病发生机制主要包括机械应力、高糖、炎症反应、氧化应激和细胞衰老导致的髓核细胞退变.目前研究发现,白藜芦醇可通过影响椎间盘髓核细胞自噬、凋亡、衰老及细胞外基质表达,达到保护及修复髓核细胞的目的,因此白藜芦醇在椎间盘退变性疾病中的作用值得研究.该文就白藜芦醇对椎间盘退变性疾病中髓核细胞的保护作用及其作用机制进...     

大鼠增龄过程中髓核组织Beclin-1和微管相关蛋白轻链3的表达及其意义

目的 探讨在增龄过程中SD大鼠髓核组织中Beclin-1和微管相关蛋白轻链3(MAPLC3)的表达及其意义.方法 3个月龄、12个月龄、24个月龄SD大鼠各8只,建立青年组、中年组和老龄组大鼠模型,苏木素-伊红(HE)染色检测椎间盘组织的退变;透射电镜检测髓核细胞中自噬体的表达;免疫组织化学染色和逆转录-聚合酶链反应(RT-PCR)法测定髓核组织中Beclin-1和MAPLC3的表达.结果 大鼠增龄过程可较好地体现椎间盘的退变变化;不同年龄组大鼠髓核细胞中均有自噬体的表达;Beclin-1和MAPLC3在不同年龄组SD大鼠髓核细胞中均有表达(0.42±0.05,0.47±0.06,0.52±0.05;0.34±0.06,0.39±0.05,0.46±0.09),且老龄组表达均较青年组高(P<0.01).Beclin-1和MAPLC3 mRNA在不同年龄组SD大鼠髓核组织中均有表达(0.86±0.12,0.93±0.14,1.01±0.13;1.09±0.06,1.15±0.07,1.33±0.11),且老龄组表达较青年组表达明显增高(分别为P<0.05和P<0.01).结论 自噬存在于椎间盘退变过程中,且MAPLC3与Beclin-1在椎间盘退变过程中的表达增加,提示自噬活性的增加可能与椎间盘退变的发展进程有关.     

退变腰椎间盘细胞经体外转基因培养生物学活性的实验研究

目的构建转化生长因子（transforming growth factor,TGF-β3的真核表达载体pEGFP-TGF-β3转染椎间盘髓核细胞,研究转基因TGF-β3对退变髓核细胞生物学特性的影响。方法通过手术方法制作椎间盘突变模型,从而获取原代退变椎间盘髓核细胞,通过脂质体将真核载体pEGFP-TGF-β3导入髓核细胞,然后对细胞的形态和增殖活性（MTT法）进行观察,应用Westen Blot检测TGF-β3在髓核细胞的表达含量,应用免疫细胞化学方法检测转染后髓核细胞的Ⅱ型胶原的表达。结果髓核细胞转染后,细胞活性增强,TGF-β3表达增加,并随着时间的延长而增加。Ⅱ型胶原表达增加。结论TGF-β3转染退变髓核细胞可起到维持髓核细胞表型,并在细胞传代后仍发挥调节作用。TGF-β3确实具有促进髓核细胞增殖和Ⅱ型胶原合成的能力,从而有可能延缓甚至逆转椎间盘退变。     

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.     

退变腰椎间盘髓核组织中NDRG2表达的变化及其意义

[目的]研究退变的腰椎间盘髓核组织中NDRG2表达的变化及其在椎间盘退变中的作用。[方法]收集腰椎间盘标本47例,根据Pfirrmann分级分为Ⅰ~Ⅴ级。采用免疫组织化学染色、实时-定量PCR(RT-PCR)和Western-Blotting技术从细胞、蛋白和基因水平分别检测腰椎间盘髓核组织中NDRG2的表达,并与Pfirrmann分级进行相关性分析;通过RT-PCR分析P53 mRNA的表达;采用衰老相关的β-半乳糖苷酶(SA-β-gal)实验检测腰椎间盘髓核组织中衰老髓核细胞比例,并与NDRG2表达量进行相关性分析。[结果]免疫组织化学染色、RT-PCR及Western-Blotting检测示NDRG2表达随椎间盘退变程度加重而增加,且与Pfirrmann分级呈正相关;P53 mRNA在腰椎间盘髓核组织中的相对表达随着椎间盘退变程度加重而增加,且与NDRG2的表达量呈正相关。SA-β-gal阳性细胞比例在椎间盘髓核组织中随退变程度加重而增加,并且与NDRG2阳性细胞比例呈正相关。[结论]NDRG2参与了腰椎间盘退变的病理过程,并且可能通过介导腰椎间盘髓核细胞衰老对椎间盘退变起促进作用。     

白介素-1β对髓核细胞MMP-1、2、9、13表达的影响

目的探讨IL-1β对人椎间盘髓核细胞表达基质金属蛋白酶MMP-1、2、9、13的作用。方法分离人椎间盘髓核细胞进行单层培养并利用甲苯胺蓝、番红O染色和Ⅱ型胶原免疫细胞化学染色进行鉴定,而后分别用10ng／ml和50ng／ml重组人IL—1β刺激体外培养的髓核细胞,RT-PCR检测基质金属蛋白酶-1、2、13的表达,定量PCR检测基质金属蛋白酶-9的表达。结果10ng／ml和50ng／ml重组人IL-1β均可促进髓核细胞基质金属蛋白酶-1、2、9、13的表达（P〈0．05）;基质金属蛋白酶-9、13表达随IL-1β浓度升高而升高（P〈0．05）。结论IL—1β可以促进人椎间盘髓核细胞表达基质金属蛋白酶-1、2、9、13,其加速了椎间盘基质分解的作用亦可能通过上述细胞因子的介导。     

营养剥夺诱导髓核细胞Bcl-2/腺病毒干扰蛋白3表达及线粒体转位的实验研究

目的通过营养剥夺模拟体内髓核细胞退变微环境,检测Bcl-2/腺病毒干扰蛋白3（Bcl-2/adenovirusE1B 19-kDa-interacting protein 3,BNIP3）表达及线粒体转位情况,为进一步探索髓核细胞退变死亡机制提供实验依据。方法成年清洁级SD大鼠2只,雌雄不限,体重150～200 g。体外分离获取鼠尾椎问盘髓核细胞,将传代后细胞分别置入正常环境（对照组:L-DMEM培养基、10%FBS、21%O₂）和营养剥夺环境（实验组:DMEM无糖无血清培养基、1%O₂）培养24、48、72 h后,实时荧光定量PCR、细胞免疫荧光染色及Western blot检测BNIP3基因及蛋白表达,流式细胞仪检测凋亡率及线粒体膜电位。结果实时荧光定量PCR、细胞免疫荧光染色及Western blot检测显示对照组细胞低表达BNIP3;实验组随培养时间延长,BNIP3表达呈上升趋势,且BNIP3与线粒体相结合;除培养后24 h实验组BNIP3基因表达与对照组比较差异无统计学意义（P>0.05）外,其余各时间点实验组BNIP3基因及蛋白表达与对照组比较差异均有统计学意义（P<0.05）。流式细胞仪检测显示,对照组细胞凋亡率较低,且细胞保持较高的线粒体膜电位;而实验组随培养时间延长细胞凋亡率增加、线粒体膜电位降低,与对照组比较差异均有统计学意义（P<0.05）。结论营养剥夺可能通过诱导BNIP3表达增加并结合线粒体导致线粒体功能障碍,最终导致髓核细胞死亡。     

Localization of bone morphogenetic protein 13 in human intervertebral disc and its molecular and functional effects in vitro in 3D culture

Our laboratory has demonstrated that bone morphogenetic protein 13 prevented the effects of annular injury in an ovine model, maintaining intervertebral disc height, cell numbers and increasing extracellular matrix production compared to degenerated controls. The present study sought to examine the molecular effects of bone morphogenetic protein 13 on human degenerated disc cells and localize its expression in both human degenerate and scoliotic disc tissue. Effect of bone morphogenetic protein 13 on human derived nucleus pulposus, annulus fibrosus and endplate cells cultured in alginate beads was evaluated by changes in proteoglycan and collagen content. Migratory potential of disc cells towards bone morphogenetic protein 13 was also examined. Bone morphogenetic protein 13 induced significant proteoglycan accumulation in nucleus (18%), annulus (21%) and endplate (23%) cells cultured in alginate beads (p < 0.05) compared to controls. Further bone morphogenetic protein 13 increased collagen I and II protein expression in nucleus and endplate cells. Nucleus cells displayed a significant chemotactic response towards bone morphogenetic protein 13. The endogenous expression of bone morphogenetic protein 13 in degenerate disc tissue was not different to scoliotic disc. Bone morphogenetic protein 13 has the potential to enhance extracellular matrix accumulation and induce cell migration in certain disc cells. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1769–1775, 2015.     

兔腰椎间盘髓核细胞的培养及形态观察

目的：通过对兔腰椎间盘髓核细胞的培养,观察细胞内的演变,探讨髓核细胞的生物学行为及影响因素。方法：取兔腰椎间盘髓核细胞,在加10％灭活胎牛血清的F12－DMEM液中培养,建立体外髓核细胞培养模型。通过光镜、电镜观察,同时进行细胞活力测定。结果：（1）原代细胞生物学性状最接近体内细胞,传代后细胞呈现衰老现象。（2） 活力测定提示随着培养时间的延长及传代,活力逐渐降低。（3）髓核细胞内细胞器的变化与其生物学活性变化相一致。结论：兔腰椎髓核细胞的体外培养成功为人腰椎髓核细胞的培养奠定了基础。     

Upregulation of BNIP3 and translocation to mitochondria in nutrition deprivation induced apoptosis in nucleus pulposus cells

ObjectiveThis study was to detect the expression of Bcl-2/adenovirus E1B19-kDa-interacting protein 3 in apoptosis induced by nutrition deprivation in nucleus pulposus cells, so as to further understand the mechanism of apoptosis in nucleus pulposus cells.MethodsCells isolated from rat caudal disc were cultured under two different oxygen, glucose and serum concentrations for up to 3 days. Interactions between two different concentrations were examined by cell vitality assay mitochondrial membrane potential (Δψm) test and apoptosis detect. The expression and location of Bcl-2/adenovirus E1B19-kDa-interacting protein 3 were tested by real-time polymerase chain reaction and immunofluorescence staining.ResultCell vitality and mitochondrial membrane potential (Δψm) were significantly reduced in absence of oxygen, glucose and serum while the cell apoptosis percent was significantly increased, as compared with the cells in normal oxygen, glucose and serum concentration. The expression of Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 showed a significant increase in absence of oxygen, glucose and serum, especially in 72 h. Furthermore, the protein was found to translocate to mitochondria.ConclusionUpregulation of Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 and translocation to mitochondria may be involved in apoptosis of nucleus pulposus cells in nutrition deprivation.     

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。     

缺氧条件下大鼠髓核细胞中缺氧诱导因子1α与自噬相关分子的表达及相关性分析

目的探讨缺氧条件下大鼠髓核细胞中缺氧诱导因子1α(hypoxia inducible factor 1α,HIF-1α)与自噬相关分子Beclin1、LC3B的表达及其相关性。方法取健康成年SD大鼠髓核组织,分离培养髓核细胞并传代。取第3代髓核细胞行HE染色和Ⅱ型胶原酶免疫荧光染色鉴定后,随机分为4组。A组细胞于常氧条件下(37℃、5%CO2、20%O2)培养,B组细胞于缺氧条件下(37℃、5%CO2、1%O2)培养,C组细胞转染HIF-1α-小干扰RNA后于缺氧条件下培养,D组细胞加入自噬抑制剂3-MA后于缺氧条件下培养。各组细胞培养8 h后,采用Western blot和实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)检测HIF-1α与自噬相关分子Beclin1、LC3B的表达情况。结果经分离纯化的第3代大鼠髓核细胞HE染色后细胞质呈淡粉色,细胞核呈蓝黑色;Ⅱ型胶原酶免疫荧光染色为阳性。Western blot和qRT-PCR检测示,B组HIF-1α、Beclin1和LC3B蛋白及mRNA相对表达量均显著高于A组(P<0.05),C组均显著低于B组,差异均有统计学意义(P<0.05)。D组HIF-1α蛋白和mRNA相对表达量与B组比较差异无统计学意义(P>0.05),Beclin1和LC3B蛋白和mRNA相对表达量均较B组显著降低(P<0.05)。结论缺氧条件能诱导大鼠髓核细胞中HIF-1α和自噬相关分子Beclin1、LC3B的表达,且HIF-1α与自噬相关分子的表达具有相关性,即HIF-1α下调能降低自噬相关分子的表达,而缺氧条件下自噬水平下调对HIF-1α的表达无明显影响。     

Anti-apoptotic effects of caspase inhibitors on rat intervertebral disc cells

BACKGROUND: Apoptosis is thought to be a critical component of disc degeneration. Two main pathways of Fas-mediated apoptosis have been identified: Type I, which is the death-inducing signaling complex pathway, and Type II, which is the mitochondrial pathway. The apoptotic pathway for anulus fibrosus cells, which is phenotypically different from that of nucleus pulposus cells, has not been elucidated to our knowledge. The ultimate initiators or executioners of apoptosis are caspases. There are also inhibitors of caspases, which have the potential of being used as anti-apoptotic therapeutic agents. We therefore undertook this study to determine (1) the apoptotic pathway of anulus fibrosus cells and (2) the anti-apoptotic potential of caspase inhibitors. METHODS: Rat anulus fibrosus cells were isolated, cultured, and placed in either 0% (apoptosis-promoting condition) or 10% (normal control) fetal bovine serum. We identified and quantified the presence of apoptotic cell death, caspase activities, and loss of mitochondrial membrane potential. In addition, we examined the cells for the expression of Fas, procaspases, and cytochrome-c. Finally, we analyzed the degree of anti-apoptotic effects of caspase inhibitors on the cells in 1% fetal bovine serum. RESULTS: The percentage of apoptosis and Fas expression in the cells incubated in 0% fetal bovine serum were increased compared with those in the cells incubated in 10% fetal bovine serum (both p < 0.001). Caspase-8, 9, and 3 activities were increased and expression of procaspases was decreased in the 0% fetal bovine serum compared with those in the 10% fetal bovine serum (all p < 0.001). In contrast, the loss of mitochondrial membrane potential and cytochrome-c release into the cytosol were unchanged in the 0% fetal bovine serum. Pancaspase and caspase-8 inhibitors reduced apoptotic cell death (p < 0.001 and p < 0.05, respectively), but caspase-9 inhibitor did not reduce apoptotic cell death. CONCLUSIONS: Our results suggest that, unlike nucleus pulposus cells, anulus fibrosus cells are Fas Type-I cells, which undergo apoptosis through the death-inducing signaling complex. We also found that apoptosis of intervertebral disc cells can be attenuated by caspase inhibitors.