人髁突骨髓间充质干细胞体内成骨的实验研究

目的 探讨人髁突来源骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSC)体内分化成骨的能力,为构建组织工程髁突提供种子细胞.方法取切除的人髁突冲洗收集骨髓细胞,采用密度梯度离心和贴壁培养法进行培养和纯化BMSC,取第3或4代BMSC进行成骨细胞和成软骨细胞诱导分化后接种于珊瑚骨支架表面,扫描电镜观察细胞在支架表面的黏附和增殖状况.将成骨或成软骨细胞-珊瑚骨支架植入裸鼠背部皮下,6和9周后观察体内成骨和成软骨情况.结果 培养3～7d后扫描电镜显示细胞黏附于珊瑚骨支架表面,呈多层生长,并跨越微孔连成网状或片状;植入裸鼠体内9周,髁突形珊瑚骨支架均基本维持最初的形态,可见散在或片状的新生骨形成,新生软骨呈岛状分布.结论从人髁突骨髓中分离出的BMSC具有体内形成新骨和软骨组织的能力,可作为构建组织工程髁突的种子细胞.     

白细胞介素-1β对关节软骨细胞线粒体和细胞内活性氧影响的研究

目的 研究重组大鼠白细胞介素-1β (rrIL-β)诱导关节软骨细胞退变是否与其破坏线粒体的完整性和促进细胞内活性氧的表达有关.方法 体外分离、培养大鼠软骨细胞成功后,以10 ng/ml白细胞介素(IL)-1β刺激,培养24 h后,通过Annexin V-异硫氰酸荧光素(FITC)和碘化丙啶双标及Hoechst 33342荧光染色检测细胞凋亡;以罗丹明-123荧光染色和活性氧检测试剂盒检测线粒体膜电位及细胞内活性氧浓度;用荧光素酶反应检测线粒体合成三磷酸腺苷(ATP)能力来评价线粒体功能.采用t检验进行统计分析.结果 IL-1β刺激组软骨细胞的线粒体膜电位、ATP含量及软骨细胞内活性氧浓度分别为(24±4)U,(4.1 ±0.8) pmol/106个细胞及(89±7)U,均低于对照组[分别为(86±10)U,(13.3±3.0) pmol/106个细胞及(17±3)U],差异均有统计学意义(t值分别为2.693,2.587,2.665,P值分别为0.026,0.039,0.021).IL-1β刺激能明显降低软骨细胞的线粒体膜电位及其合成ATP的能力,并且能促进细胞内活性氧的表达.结论 IL-Iβ能通过破坏软骨细胞线粒体膜的完整性和促进细胞内活性氧的表达,来诱导软骨细胞去分化,从而促进关节软骨退变.     

周期性单轴压力对大鼠髁突软骨细胞结缔组织生长因子表达的影响

目的 探讨在髁突软骨细胞受到周期性单轴压力后,结缔组织生长因子(connective tissuegrowth factor, CTGF)表达的影响,为正畸治疗中髁突软骨受力后的改建提供生物学依据。方法 选取1周龄SD大鼠,提取并培养髁突软骨细胞,免疫组化鉴定。利用四点弯曲细胞力学加载仪对第3代细胞进行力值为2000u strain、0.5Hz的体外周期性单轴压力加载,分别在加力0min、30min、60min和120min后继续培养24h,应用蛋白印迹法检测在不同加力时间CTGF蛋白表达的变化。应用SPSS18.0软件对数据进行统计学分析。结果 CTGF的相对蛋白量在加力0min、30min、60min和120min后,分别为0、1.59、2.34和3.16,随着加载时间的增加,表达呈逐渐上升趋势。且组间差异具有统计学意义(P<0.05) 结论 周期性单轴压力可刺激大鼠髁突软骨细胞CTGF的表达。     

体外周期性单轴压应力下髁突软骨细胞Raf激酶抑制蛋白(RKIP)的动态变化研究

目的:探讨体外周期性单轴压应力对大鼠髁突软骨细胞的Raf激酶抑制蛋白(RKIP)及其mRNA变化的早期影响,以助于阐明力学引起细胞应答反应的分子机制。方法:利用四点弯曲细胞力学加载仪对第3代大鼠髁突软骨细胞进行体外周期性单轴压应力加载,力值为4000μstrain,时间为0、15、30、60、120、240min。采用实时荧光定量PCR技术和Western blot免疫印迹技术研究大鼠髁突软骨细胞在周期性单轴压应力作用下RKIP及其mRNA变化。结果:大鼠髁突软骨细胞RKIP mRNA和蛋白表达呈现几乎相反的趋势。RKIP mRNA的表达于30min上升(P<0.001),60min达到高峰后迅速回落,低于细胞的正常基态水平;而RKIP的蛋白表达30min开始下调(P<0.001),持续至120、240min时回升。结论:RKIP在髁突软骨细胞的早期力学应答机制中可能扮演着相当重要的作用,其蛋白和mRNA水平表达变化不同,转录水平的调控最终要影响蛋白水平的表达。     

Effect of heat stimulation on viability and proteoglycan metabolism of cultured chondrocytes: preliminary report

 Thermotherapy has been applied to various joint diseases and injuries, but its direct effects on articular cartilage have remained unclear. The present study examined the effects on cell viability and metabolism by using the chondrocyte-like cell line HCS-2/8. The temperatures and durations of heat stimulation were 39°, 41°, 43°, and 45°C for 15 or 30 min. After heat stimulation of 41°C or lower for 15 or 30 min, cell viability increased and proteoglycan metabolism was accelerated, whereas after stimulation at 43°C or higher for 30 min the viability and metabolism decreased. These results indicate that appropriate heat stimulation positively affects cell viability and the proteoglycan metabolism of articular cartilage, whereas too much heat stimulation produces negative effects. Clinical efficacy is therefore determined by the overall thermal dose. When the appropriate combination of temperature and duration is found, thermotherapy for diseases and injury of articular cartilage can be highly useful in clinical practice. Received: July 26, 2002 / Accepted: December 24, 2002 RID="*" ID="*" Offprint requests to: T. Hojo     

Statin stimulates bone morphogenetic protein-2, aggrecan,and type 2 collagen gene expression and proteoglycan synthesis in rat chondrocytes

Statins increase bone morphogenetic protein-2 (BMP-2) mRNA expression and subsequently increase new bone formation in vitro. However, the action of statins on the BMP-2 mRNA regulation of cartilage matrix synthesis by chondrocytes is unknown. We evaluated regulation of BMP-2, aggrecan, and type II collagen (COL2) mRNA and ³⁵S-labeled proteoglycan (PG) synthesis by mevastatin using cultured chondrocytes obtained from articular cartilage of fetal rats. Expression of BMP-2, aggrecan, and COL2 mRNAs were increased in the presence of 2µM mevastatin on day 2. However, longer (10 day) culture in the presence of the drug decreased the expression of these mRNAs. PG synthesis was increased 3 days after treating the cells with mevastatin, which was also decreased with longer (10 day) mevastatin treatment. These results suggest that mevastatin increases mRNA expression of BMP-2, aggrecan, and COL2 as well as PG synthesis by fetal rat chondrocytes early in the treatment period. We suggest that statins have implications for fracture and cartilage repair.     

In vitro study of the antioxidant properties of nimesulide and 4-OH nimesulide: effects on HRP- and luminol-dependent chemiluminescence produced by human chondrocytes

OBJECTIVES: Reactive oxygen species (ROS) are now recognized to play an important role in the pathogenesis of rheumatic diseases and constitute an interesting therapeutic target for drugs. This in vitro study was designed to evaluate the antioxidant properties of nimesulide (NIM), a nonsteroidal antiinflammatory drug of the sulfonanilide class, and its main metabolite 4-OH nimesulide (4-OHNIM). METHODS: The scavenging effects of NIM and 4-OH NIM on hydroxyl radical ((.)OH) and superoxide anions (O(minusd)(2)) were investigated by electron spin resonance (ESR), using 5, 5-dimethylpyrroline-N-oxide (DMPO) as the spin trap agent. The quenching properties of these drugs on hypochlorite anion was studied by luminol enhanced chemiluminescence. Finally, the effects of NIM and 4-OHNIM on the reactive oxygen species production by human articular chondrocytes were recorded by HRP and luminol-enhanced chemiluminescence. RESULTS: By this method it has been demonstrated that NIM and 4-OHNIM, at concentrations ranging from 10 to 100 microM, are potent scavengers of(.)OH whereas only 4-OHNIM was capable to scavenge O(minusd)(2). Chemiluminescence generated by HOCl was also significantly and dose-dependently inhibited by both NIM and 4-OHNIM. Nevertheless, at each concentration tested, the inhibitory effect of 4-OHNIM was significantly more marked, even at the highest concentration (100 microM). Furthermore, when chondrocytes were pre-incubated for 48-96 h with NIM or 4-OHNIM, the luminol- and HRP-dependent CL produced by the cells was significantly inhibited in a dose-dependent manner. CONCLUSIONS: NIM and 4-OHNIM may protect cartilage against oxidative stress, not only by scavenging ROS but also by inhibiting their production by chondrocytes.     

透明质酸对人骨关节炎软骨细胞保护机制的探讨

目的:探讨透明质酸对人骨关节炎软骨细胞的作用机制。方法:应用细胞培养、四唑盐比色实验检测细胞线粒体活性,流式细胞术检测线粒体膜电位、细胞凋亡百分率和细胞内游离钙离子浓度。结果:透明质酸能明显提高人骨关节炎软骨细胞线粒体活性,抬高线粒体膜电位,降低细胞凋亡率和胞内游离钙浓度。结论:透明质酸可抑制人骨关节炎软骨细胞线粒体膜电位的降低,从而具有稳定线粒体膜电位的作用,抑制细胞凋亡的发生,这种作用可能与其能抑制软骨细胞内钙超载有关。     

Novel hyaluronic acid-chitosan nanoparticles as non-viral gene delivery vectors targeting osteoarthritis

Gene therapy is a promising new treatment strategy for common joint-disorders such as osteoarthritis. The development of safe, effective, targeted non-viral gene carriers is important for the clinical success of gene therapy. The present work describes the use of hybrid hyaluronic acid (HA)/chitosan (CS) nanoparticles as novel non-viral gene delivery vectors capable of transferring exogenous genes into primary chondrocytes for the treatment of joint diseases. HA/CS plasmid-DNA nanoparticles were synthesized through the complex coacervation of the cationic polymers with pEGFP. Particle size and zeta potential were related to the weight ratio of CS to HA, where increases in nanoparticle size and decreases in surface charge were observed as HA content increased. The particle size and the zeta potential varied according to pH. Transfection of primary chondrocytes was performed under different conditions to examine variations in the pH of the transfection medium, different N/P ratios, different plasmid concentrations, and different molecular weights of chitosan. Transfection efficiency was maximized for a medium pH of approximately 6.8, an N/P ratio of 5, plasmid concentration of 4 μg/ml, and a chitosan molecular weight of 50 kDa. The transfection efficiency of HA/CS-plasmid nanoparticles was significantly higher than that of CS-plasmid nanoparticles under the same conditions. The average viability of cells transfected with HA/CS-plasmid nanoparticles was over 90%. These results suggest that HA/CS-plasmid nanoparticles could be an effective non-viral vector suitable for gene delivery to chondrocytes.     

Egr-1介导了IL-1β诱导的小鼠软骨细胞凋亡

目的:研究早期生长反应蛋白1(earlygrowthresponseprotein1,Egr-1)在白细胞介素1β(interleukin-1β,IL-1β)诱导的软骨细胞凋亡中的作用。方法:体外分离培养小鼠关节软骨细胞,用IL-1β刺激不同的时间后,用Hoechst33258染色,计算发生核皱缩细胞占全部细胞的比例;采用定量RT-PCR(qRT-PCR)方法检测Egr-1mRNA表达水平;采用Westernblotting方法检测Egr-1及激活型caspase-3蛋白的表达水平;采用LipofectamineRNAiMAX将Egr选择性干扰片段转染软骨细胞,采用qRT-PCR及Westernblotting观察RNA片段的干扰效率。结果:IL-1β刺激体外培养的软骨细胞4h、8h和12h后,分别引起约7%、22%和41%细胞凋亡,同时激活型caspase-3表达上调。IL-1β诱导软骨细胞凋亡的同时,Egr-1mRNA及蛋白质呈时间依赖的表达上调。2个siRNA片段成功干扰Egr-1表达后,细胞12h凋亡率从约40%下降到14%和12%,同时激活型caspase-3的表达被有效抑制。结论:Egr-1介导了IL-1β诱导的软骨细胞凋亡,可能在骨关节炎的发病过程中发挥重要作用。