芹菜素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蛋白多糖合成的影响.[方法]32例后路腰椎间盘切除术的志愿患者中获取新鲜的32个椎间盘样品,每1例样品被切成1～2 mm3大小的碎块,与1ml培养基DMEM一同装入2.5ml的注射器中,放入培养椎间盘组织的压力装置.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 (SNAP),在可调静水压装置中培养2h后提取上清液,采用考马斯亮蓝蛋白定量检测法测定上清液中蛋白多糖的含量.[结果]芹菜素3atm组和L- NMMA3atm组蛋白多糖合成最高,两组间相比无显著性差异(P＞0.05),与空白组相比有显著性差异(P＜0.05),SNAP 30 atm组蛋白多糖合成最低,与各组间相比均有显著性差异(P＜0.01).空白组、L-NMMA组、SNAP组和芹菜素组中3 atm下蛋白多糖合成较30 atm下蛋白多糖合成高,与之相比有显著性差异(P＜0.05).[结论]3 atm的静水压下,培养基中添加芹菜素使蛋白多糖合成明显增加.添加芹菜素也部分降低了 30atm的静水压下对蛋白多糖合成的抑制.     

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.     

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     

一氧化氮在腰椎间盘退变中的作用

目的：探讨一氧化氮（ＮＯ）在腰椎间盘退变中的作用。方法 随机选择15只SD大鼠分为实验组和对照组,实验组采用切除后柱法建立腰椎间盘退变模型。结果 3周后处死大鼠摄动力位X线片并取椎间盘测定NO、IL－6,实验组较对照组NO含量明显升高（P＜0．05,P＜0．01）,而且实验组中应用精氨酸者较非应用精氨酸者腰椎退变不稳和骨赘增生的程度要轻。IL－6对照组较实验组较高,但无统计学意义（P＞0．05）。结论 腰椎退变过程中NO的合成增加有助于延缓椎间盘的退变,IL－6同椎间盘的退变无明显的相关性。     

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。     

腺相关病毒载体介导的TIMP1对人退变腰椎间盘髓核细胞蛋白多糖的影响

目的:探讨腺相关病毒(Adeno-associated virus;AAV)载体介导的基质金属蛋白酶组织抑制剂1(tissue inhibitor of metalloproteinases 1;TIMP1)对人退变腰椎间盘髓核细胞的生物学效应.方法:单层培养并鉴定人退变腰椎间盘髓核细胞.采用绿色荧光蛋白标记的腺相关病毒(rAAV2-EGFP)检测其对髓核细胞的转染效率.应用构建的rAAV2-TIMP1转染髓核细胞;通过细胞形态学观察、35S标记氨基酸整合法检测rAAV2-TIMP1对人退变腰椎问盘髓核细胞基质合成的影响.在35S检测中;以未转染的退变髓核细胞做为正常对照组.结果:光镜下观察所培养的细胞类型以成纤维样细胞为主;Ⅱ型胶原免疫组化和番红O染色鉴定培养细胞为髓核细胞.AAV转染人退变腰椎间盘髓核细胞的转染效率为12%.35S标记整合法检测rAAV2-TIMP1转染组每分钟计数值为341.43±42.85;正常对照组为224.20±29.26;两组间比较差异有统计学意义(P＜0.05).结论:TIMP1能够促进退变椎间盘髓核细胞蛋白多糖的合成.     

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     

一氧化氮对腹膜间皮细胞表面蛋白-A表达的影响

目的：探讨在体外一氧化氮对腹膜间皮细胞表面蛋白-A（SP-A）mRNA和蛋白表达的影响。方法：Ⅱ级SD雄性大鼠,腹腔注射25ml0.25%胰蛋白酶消化液后,2h后处死大鼠,获取的腹膜间皮细胞用DMEM/F12培养液（含10%FBS）培养于6孔培养板中,待细胞传致第3代时,加入不同浓度的SNAP（nirtic oxide donor）分为：正常对照组、0.6μmol/LSNAP组、0.9μmol/L SNAP组、1.2μmol/L SNAP组、2.4μmol/L SNAP组,每个组6孔,培养24h后提取细胞RNA及蛋白;另外,将1.2μmol/L SNAP与腹膜间皮一起培养,分别于0.5h、1h、2h、4h、8h、12h、24h及48h时提取细胞RNA及蛋白。分别用RT-PCR及Western blot检测腹膜间皮细胞SP-A mRNA的表达和蛋白的合成。结果：与正常对照组相比,SNAP能抑制SP-A mRNA的表达和蛋白的合成（P〈0.05）,且随着SNAP浓度的增高而增加,当SNAP浓度为1.2μmol/L时,随着培养时间的增加,SP-A mRNA的表达和蛋白的合成也逐渐被抑制。结论：一氧化氮能抑制腹膜间皮细胞SP-AmRNA的表达和蛋白的合成,且呈剂量及时间依赖性。     

腰椎间盘蛋白多糖核心蛋白的基本表达

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ｍＲＮＡ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     

白细胞介素1α对椎间盘蛋白多糖代谢的影响

目的 研究白细胞是介素１α（ｉｎｔｅｒｌｅｕｋｉｎ－１α,ＩＬ－１α）对椎商盘髓核中蛋白多糖代谢的影响。方法 取自然流产的胎儿１具,胎龄８个月,术后４小时内无菌取出椎间盘,进行髓核组织块的体外培养,在培养液中分别加入ＩＬ－１α０．１、０．２、０．４ｎｍｏｌ／Ｌ,培养３０小时后,用Ａｌｃｉａｎ法检测培养液中踱酸软骨素的含量。在髓核组织培养液中加入ＩＬ－１α ０．３ｍｍｏｌ／Ｌ,分别培养１２、２４、７     

一氧化氮合酶抑制剂对延缓腰椎间盘退变的影响

目的　探讨一氧化氮合酶(NOS)抑制剂L N6 亚氨乙基 赖氨酸(L NIL)和S 甲基异硫脲(SMT)对退变腰椎间盘组织代谢的影响。方法　无菌条件下,取2 0例腰椎间盘突出症患者的椎间盘组织体外培养,分别加入1mmol/L浓度的SMT和L NIL ,培养72h后,通过检测硝酸盐和亚硝酸盐的含量来观察椎间盘NO的释放量及NOS的活性;原位杂交法检测椎间盘组织iNOSmRNA和MMP3mRNA的表达。培养10d后,化学比色法观察椎间盘蛋白多糖含量和羟脯氨酸释放量的变化。结果　L NIL组髓核和纤维环NO释放量(65 .6±4.5 ,68.8±5 .7) μmol/L和SMT组髓核和纤维环NO释放量(69.5±6.5 ,69.1±6.1) μmol/L较对照组NO释放量(10 7.9±4.4,93 .1±5 .9) μmol/L明显减少(P <0 .0 1)。L NIL组和SMT组髓核组织中蛋白多糖含量(5 1.3±9.6,48.2±8.5 )kg/L ,比对照组(3 2 .1±6.4)kg/L明显增加(P <0 .0 1) ,羟脯氨酸释放量(1.1±0 .4,1.2±0 .5 )kg/L比对照组(3 .4±0 .8)kg/L显著减少(P <0 .0 1) ;同时,原位杂交法未检测到iNOSmRNA和MMP3mRNA的表达。结论　NOS抑制剂L NIL和SMT能抑制过量NO的释放,对延缓椎间盘退变具有积极的作用     

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     

Effect of dynamic hydrostatic pressure on rabbit intervertebral disc cells.

The pathogenesis of vibration-induced disorders of intervertebral disc at the cellular level is largely unknown. The objective of this study was to establish a method to investigate the ranges of constructive and destructive hydrostatic loading frequencies and amplitudes in preventing or inducing extracellular disc matrix degradation. Using a hydraulic chamber, normal rabbit intervertebral disc cells were tested under dynamic hydrostatic loading. Monolayer cultures of disc outer annulus cells and 3-dimensional (3-D) alginate cultures of disc nucleus pulposus cells were tested. Effects of different loading amplitudes (3-D culture, 0-3 MPa; monolayer, 0-1.7 MPa) and frequencies (1-20 Hz) on disc collagen and protein metabolism were investigated by measuring 3H-proline-labeled proteins associated with the cells in the extracellular matrix and release of 3H-proline-labeled molecules into culture medium. High frequency and high amplitude hydrostatic stress stimulated collagen synthesis in cultures of outer annulus cells whereas the lower amplitude and frequency hydrostatic stress had little effect. For the same loading duration and repetition, neither treatment significantly affected the relative amount of protein released from the cell layers, indicating that protein degradation and stability were unaffected. In the 3-D nucleus culture, higher amplitude and frequency increased synthesis rate and lowered degradation. In this case, loading amplitude had a stronger influence on cell response than that of loading frequency. Considering the ranges of loading amplitude and frequency used in this study, short-term application of high loading amplitudes and frequencies was beneficial in stimulation of protein synthesis and reduction of protein degradation.     

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。     

一氧化氮在突出腰椎间盘中的表达及其意义

目的 :研究一氧化氮 (NO)在突出腰椎间盘组织中的含量及组织学定位 ,并对其意义进行探讨。方法 :对 32例腰椎间盘突出患者的突出间盘组织采取两种方法进行研究 :(1) 12例做体外培养 ,用分光光度法测定培养液上清中NO含量 ;(2 ) 2 0例用免疫组化方法对产生NO的细胞类型及组织学定位进行研究。同时对取自 4具新鲜尸体的 12个正常椎间盘采用相同方法做为对照。结果 :突出腰椎间盘组织产生NO的量为 2 0 0 70± 6 5 5 5nmol/g ,正常对照组的NO量为 76 31± 19 49nmol/ g ,两者统计学有显著性差异 (P <0 0 0 1)。免疫组化结果发现 ,2 0例患者椎间盘组织中一氧化氮合成酶表达阳性 16例 ,12个正常椎间盘组织中无表达阳性细胞。结论 :诱导型一氧化氮合成酶主要由突出椎间盘周围的肉芽组织产生 ,阳性细胞主要以成纤维细胞、软骨细胞及淋巴细胞为主。腰椎间盘可自身合成NO ,NO可能在椎间盘退变中起重要作用 ,突出腰椎间盘中的NO主要由突出腰椎间盘周围的肉芽组织产生。     

The effects of hydrostatic pressure on matrix synthesis in articular cartilage

The direct effects of hydrostatic pressure on matrix synthesis in articular cartilage can be studied independently of the other factors that change during loading. We have found that the influence of hydrostatic pressure on incorporation rates of 35SO4 and [3H]proline into adult bovine articular cartilage slices in vitro depends on the pressure level and on the time at pressure. Pressures in the "physiological" range (5-15 MPa) applied for 20 s or for 5 min could stimulate tracer incorporation (30-130%) during the following 2 h, but higher pressures (20-50 MPa) had no effect on incorporation rates. The degree of stimulation in cartilage obtained from different animals was found to vary; in some animals none was seen. Stimulation also varied with position along the joint. Physiological pressures (5-10 MPa) applied continuously for the 2-h incubation period also stimulated incorporation rates, but pressures greater than 20 MPa always produced a decrease that was related to the applied pressure and that was reversible. These results suggests that the hydrostatic pressure that occurs during loading is a signal that can stimulate matrix synthesis rates in articular cartilage.     

Attenuation of apoptosis and enhancement of proteoglycan synthesis in rabbit cartilage defects by hyperbaric oxygen treatment are related to the suppression of nitric oxide production.

Proinflammatory cytokine, nitric oxide (NO) and localized hypoxia-induced apoptosis and proteoglycan (PG) degradation are thought to be correlated to the degree of cartilage injury. This study evaluated hyperbaric oxygen (HBO)-induced changes in joint cavity oxygen tension, antigenickeratan sulfate (KS) content, inducible nitric oxide synthase (iNOS) expression, PG synthesis, and cell apoptosis in full-thickness defects of rabbit cartilage. The HBO group was exposed to 100% oxygen at 2.5 atm for 2 h daily, 5 days per week. Meanwhile, the control group was kept in housing cages with normal air. The joint cavity oxygen tension was determined with an oxygen sensor. Blood serum KS was quantified by competitive indirect enzyme-linked immunosorbent assay (ELISA). After sacrifice, specimen sections were sent for histological and histochemical examination with a standardized scoring system. In situ analysis of iNOs expression and apoptosis detection were performed using immunostaining and TUNEL staining, respectively and quantified by a computerized imagine analysis system. This study demonstrated that HBO treatment increased joint cavity oxygen tension but decreased blood KS content. Histological and histochemical score results showed that HBO treatment significantly increased the cartilage repair. Moreover, immunostaining and TUNEL staining showed that HBO treatment suppressed the iNOs expression and apoptosis of chondrocytes, respectively. Accordingly, HBO offers a potential treatment method for cartilage injury.     

Temporo-spatial distribution of blood vessels in human lumbar intervertebral discs

While there is consensus in the literature that blood vessels are confined to the outer anulus fibrosus of normal adult intervertebral disc, debate continues whether there is a vascular in-growths into inner parts of the intervertebral disc during degeneration. We therefore tested the hypothesis that vascular in-growth is not a distinct feature of disc degeneration. The specific endothelial cell marker CD 31 (PECAM) was used to immunohistochemically investigate 42 paraffin-embedded complete mid-sagittal human intervertebral disc sections of various ages (0–86 years) and varying extent of histomorphological degeneration. Additionally, 20 surgical disc samples from individuals (26–69 years) were included in this study. In discs of fetal to infantile age, blood vessels perforated the cartilaginous end plate and extended into the inner and outer anulus fibrosus, but not into the nucleus pulposus. In adolescents and adults, no blood vessels were seen except for the outer zone of the anulus fibrosus adjacent to the insertion to ligaments. The cartilaginous end plate remained free of vessels, except for areas with circumscribed destruction of the end plate. In advanced disc degeneration, no vessels were observed except for those few cases with complete, scar-like disc destruction. However, some rim lesions and occasionally major clefts were surrounded by a small network of capillary blood vessels extending into deeper zones of the anulus fibrosus. A subsequent morphometric analysis, revealed slightly “deeper” blood vessel extension in juvenile/adolescent discs when compared to young, mature and senile adult individuals with significantly “deeper” extension in the posterior than anterior anulus. The analysis of the surgical specimens showed that only sparse capillary blood vessels which did not extend into the nucleus pulposus even in major disc disruption. Our results show that vascular invasion deeper than the periphery was not observed during disc degeneration, which supports the hypothesis that vascular in-growth is not a distinct feature of disc degeneration. This study was supported by a grant from the AO/ASIF Foundation Switzerland (00-B72) and a grant from the AO Spine (SRN 02/103).     

一氧化氮和一氧化氮合成酶在急性肺损伤的作用

急性肺损伤(acute lung injury,ALI)是一种发病率和死亡率都很高的常见临床疾病. 目前,对ALI病理生理学基础和临床研究方面的了解越来越多,但并没有提出新的治疗策略能够明显改善ALI的治疗.在ALI的动物模型和患者中,一氧化氮合成酶(nitric oxide synthases,NOS)表达及活性增强和一氧化氮(NO)的增多在ALI的病理生理过程中有重要作用;但临床抑制NO生成以及选择性抑制NOS并没有对ALI的治疗有明显效果.目前提出了不同细胞源性NO的概念,这种NO的细胞源性差异可能对ALI的治疗有潜在的意义.现综述NO和NOS在ALI中的作用.