恒定磁场中大鼠成骨细胞的增殖和功能活性

背景：磁场对成骨细胞生物学的影响存在一定的分歧。 目的：探讨不同强度恒定磁场作用不同时间后成骨细胞增殖和功能活性的改变。 方法：用体外培养的SD大鼠成骨细胞第3~5代分别在0,5,22,86,135 mT恒定磁场下培养,观察8,12,24 h后细胞增殖和凋亡变化及细胞上清液中骨钙素及Ⅰ型前胶原C端前肽的表达。 结果与结论：磁场作用8,12 h后,5,22,86,135 mT磁场培养的细胞增殖指数均高于对照组(P < 0.05),作用24 h时,仅5 mT组高于对照组(P < 0.05)。而0,5,22,86,135 mT恒定磁场下培养8,12,24 h的凋亡百分数差异无显著性意义。磁场作用8 h后,22,86,135 mT组骨钙素分泌量均高于对照组(P < 0.05);作用24 h后,135 mT组骨钙素分泌量则明显低于对照组(P < 0.05);而磁场作用8,12 h后,22,86 mT组Ⅰ型前胶原C端前肽分泌量明显高于对照组(P < 0.05),作用24 h后,135 mT组Ⅰ型前胶原C端前肽分泌量则明显低于对照组(P < 0.05)。表明低强度磁场、短时间作用可促进成骨细胞的增殖及成骨物质的分泌,而高强度磁场或磁场暴露时间过长则抑制成骨细胞增殖及成骨物质的分泌。     

假体磨损颗粒钴铬离子影响成骨细胞增殖及RANKL、骨保护素的表达

背景：金属-金属假体置入体内后可以发生腐蚀或磨损,释放镍、钴、铬、钛等金属离子,诱导局部炎性因子的释放。 目的：观察Co2+、Cr3+对小鼠成骨细胞增殖的影响,以及成骨细胞暴露在Co2+ 、Cr3+条件下RANKL、骨保护素基因的表达。 方法：体外培养成骨细胞,实验分两组,对照组给予生理盐水,离子组给予钴、铬离子干预。 结果与结论：显微镜直接计数法显示干预后1~6 d对照组随时间推移细胞数目显著增加,离子组则增加不明显。共培养24,48 h后,RT-PCR结果显示离子组RANKL、骨保护素基因表达较对照组均增加,以RANKL增加更显著(P < 0.05),RANKL/OPG mRNA的比率也明显增加。提示金属离子对成骨细胞的增殖有显著抑制作用,且可刺激成骨细胞RANKL、骨保护素mRNA的表达。     

不同强度静磁场对成骨细胞细胞骨架改建的影响☆

背景：静磁场对成骨细胞增殖活性、细胞因子分泌及碱性磷酸酶表达等功能活性影响的机制尚不清楚。 目的：观察不同强度静磁场加载对成骨细胞骨架改建和细胞骨架蛋白表达的影响。 方法：取新生24 h内SD大鼠颅骨进行成骨细胞的培养和鉴定。采用静磁场加载装置对体外培养的成骨细胞进行0,8,50,160 mT的静磁场加载,分别于静磁场加载24,48,72 h应用BODYPY-Phaloidin进行成骨细胞骨架染色,激光扫描共聚焦显微镜和图像处理软件Image J测定细胞骨架蛋白的荧光强度。 结果与结论：8,50,160 mT静磁场加载24,48,72 h,荧光标记的成骨细胞骨架蛋白向细胞核周围集中,荧光强度增强(P < 0.05),其中经50 mT静磁场加载的成骨细胞骨架蛋白的荧光最强。说明一定强度的静磁场可以影响成骨细胞骨架的改建和重组。     

金属离子对单核/巨噬细胞细胞活性及其膜上RANK表达的影响

背景：与其他配伍的假体一样,金属-金属假体在使用过程中也会产生大量的磨损颗粒和金属离子,其中金属离子以钴和铬离子较为常见,可导致假体周围骨溶解,引起假体无菌性松动。 目的：观察Co2+、Cr3+离子对体外培养小鼠单核/巨噬细胞(RAW264.7)的细胞活性及其膜上RANK表达的影响。 方法：体外培养单核/巨噬细胞(RAW264.7),采用金属钴铬离子对单核/巨噬细胞进行干预,不同时间点用四唑盐比色方法检测细胞活性并以半定量反转录-聚合酶链反应方法测定RANK mRNA的表达量。 结果与结论：四唑盐比色实验结果显示,与对照组相比,Co2+、Cr3+可使单核/巨噬细胞的细胞活性明显下降。单核/巨噬细胞暴露在钴铬离子下,与对照组相比,钴铬离子组单核/巨噬细胞RANKmRNA在12 h表达增强(P < 0.05),24 h达到高峰(P < 0.05),48 h较24 h表达下降(P < 0.05)。结果提示,金属离子对单核/巨噬细胞有细胞毒性,且能够刺激单核/巨噬细胞RANK mRNA的表达,为单核/巨噬细胞向具有骨质吸收功能的破骨样细胞转化提供必要的前提条件。     

雌激素受体基因ERβ与小鼠成骨细胞的增殖和分化

背景：雌激素受体ERs表达于所有关系到骨形成和骨吸收的细胞成分中。 目的：观察雌激素受体ERβ对成骨细胞增殖、分化能力的调控作用。 方法：以小鼠成骨细胞株MC3T3-E1为对象,设立3组：实验组转染雌激素受体ERβ RNAi载体、阴性对照组转染ERL RNAi载体、空白对照组不进行转染,3组在相同条件下培养。采用MTT法绘制各组细胞的生长曲线;流式细胞仪分析各组细胞的细胞周期。 结果与结论：实验组成骨细胞的增殖能力明显高于空白对照组和阴性对照组,G1期细胞百分率明显少于空白对照组和阴性对照组,而S期和G2期细胞百分率明显高于空白对照组和阴性对照组(P均< 0.05)。根据雌激素受体ERβ沉默后检测的结果可以反向推断,ERβ的表达对成骨细胞的增殖、分化具有抑制作用。     

高精度快速成型模型的制作精度及其体外细胞毒性

背景：快速成型技术在手术模拟与组织工程研究的应用日益广泛,而现场手术模拟和术前指导对模型的精度要求非常高,且在术中使用模型应要求其对人体无毒性。 目的：评估不同后处理方法快速成型模型的成型精度及其细胞毒性。 方法：采用选择性激光烧结技术制作标准圆柱体样品模型,并经浸蜡或树脂处理后,测量模型的高度和底面直径,评估其成型精度。然后采用噻唑蓝法研究不同后处理的模型件对小鼠成纤维细胞L929和小鼠前成骨细胞MC3T3-E1的毒性作用。实验分为石蜡处理组、树脂处理组、阴性对照组(未处理模型)、空白对照组(新鲜培养基)及阳性对照组(体积分数5%的DMSO),培养2 d后,490 nm处测量吸光度,并计算其相对增殖率和评价细胞毒性等级。 结果与结论：浸蜡、树脂处理后的模型以及模型原件的精度为95%~97%,误差为0.5~1 mm。模型浸提液培养L929和MC3T3-E1细胞2 d后,各实验组的细胞相对增殖率均大于80%,石蜡处理后的快速成型模型对L929和MC3T3-E1有较低的毒性作用,而树脂处理和未处理的模型对这两种细胞均无毒性作用,细胞毒性均为0~1级。     

美满霉素对帕金森病细胞凋亡模型保护作用的研究

目的探讨美满霉素(MC)对1甲基4苯基吡啶离子(MPP+)诱导的帕金森病细胞凋亡模型保护作用的机制。方法将不同浓度(10、50、250、500μmol/L)MPP+加入培养的PC12细胞中,选择最适当浓度的MPP+建立多巴胺神经元凋亡模型(MPP+组);通过四甲基偶氮唑盐法(MTT法)检测经不同浓度(0、10、50、100、200μmol/L)MC预处理后多巴胺神经元凋亡模型的活性,以此筛选出具有最佳保护作用MC浓度建立MC+MPP+组;用电泳法、流式细胞术和逆转录聚合酶链式反应分别检测MPP+组、MC+MPP+组细胞的凋亡率,以及此2组细胞caspase3mRNA的表达,并与空白对照组比较。结果(1)在MPP+为10μmol/L时可见细胞凋亡最典型的梯状DNA条带,以此浓度建立多巴胺神经元凋亡模型(MPP+组);用100μmol/LMC预处理的MPP+组细胞活性最高(P<0.05),以此作为此后实验用MC浓度。(2)MC+MPP+组细胞凋亡率及caspase3mRNA的灰度比值分别为(22.83±2.10)%及68.08±1.14,极显著低于MPP+组的(45.89±2.28)%及86.50±1.43(均P<0.01),但仍明显高于空白对照组的(11.05±1.02)%及53.75±1.23(均P<0.05)。结论MC可能通过下调caspase3mRNA表达保护MPP+诱导的PC12细胞凋亡。     

碱性成纤维细胞生长因子对庆大霉素致肾小管上皮损伤的拮抗效应

背景：体内实验证实成纤维细胞生长因子能有效保护庆大霉素致肾小管上皮细胞的损伤，但对体外培养细胞的作用如何不多见。 目的：在建立庆大霉素肾毒性体外细胞模型的基础上，观察不同浓度碱性成纤维细胞生长因子对庆大霉素肾毒性的保护作用。 方法：采用酶加网筛方法分离纯化昆明小鼠肾小管上皮细胞，调整细胞浓度为1×108 L-1，将细胞悬液移入96孔细胞培养板，分组培养：空白对照组：正常培养；庆大霉素组: 10，30，50 µL/孔 (即400，1 200，2 000 U/孔)记为G1、G2、G3；碱性成纤维细胞生长因子组：20，50，80 µL/孔(即90，225，360 ng/孔)记为B1、B2、B3；庆大霉素加碱性成纤维细胞生长因子干预组：先加碱性成纤维细胞生长因子12 h后，再加庆大霉素12 h培养，分9个剂量组，即G1B1、G1B2、G1B3、G2B1、G2B2、G2B3、G3B1、G3B2、G3B3，每组4复孔。观察细胞形态及数量变化。 结果与结论：庆大霉素对肾小管上皮细胞的损伤呈剂量依赖性，中、高浓度组的上皮细胞皱缩，变圆，肿胀，贴壁差，内部胞质破坏严重，结构紊乱，低浓度组细胞数量改变不明显，并且开始有成纤维细胞出现；碱性成纤维细胞生长因子各组细胞饱满、折光性强，数量明显增多，50 µL/孔浓度以上效果显著，与80 µL/孔差异无显著性意义；庆大霉素加碱性成纤维细胞生长因子干预组中低浓度庆大霉素组细胞未见明显损害，细胞数量反而增多，中浓度庆大霉素组损害的细胞崩解减少、细胞皱缩和贴壁差的程度有所减轻，高浓度碱性成纤维细胞生长因子干预后细胞形态良好，但高浓度庆大霉素所致细胞肿胀、坏死损伤任何浓度的碱性成纤维细胞生长因子干预都无法改善。50 µL/孔碱性成纤维细胞生长因子对中、低浓度庆大霉素所致肾毒性有拮抗作用，对高浓度庆大霉素所致肾毒性无保护作用。     

镁锌合金对成骨细胞整合素β1表达的影响*☆

背景：自主设计的可降解镁锌合金既保持了镁合金与人骨接近的密度和弹性模量,又排除了商业镁合金中铝和稀土元素的毒性。 目的：观察新型可降解镁锌合金(Mg-6%Zn)对前成骨细胞MC3T3-E1细胞整合素β1表达的影响。 设计、时间及地点：细胞分子学水平,对比观察实验,于2008-03/05在上海交通大学附属第六人民医院中心实验室完成。 材料：实验可降解Mg-6%Zn合金由上海交通大学材料科学与工程学院研制,密度是1.82 g/cm3,弹性模量44 GPa,以临床常用的可吸收内植物左旋聚乳酸做为对照。MC3T3-E1细胞由中国科学院典型培养物保藏委员会细胞库提供。 方法：将MC3T3-E1细胞接种于镁锌合金和左旋聚乳酸上,共培养2,24,48 h后用扫描电镜观察细胞形态;共培养1,3,6,9,12,15 d后,收集细胞,利用实时荧光定量PCR方法检测成骨细胞整合素β1mRNA的表达水平。 主要观察指标：MC3T3-E1细胞在材料表面生长形态和整合素β1mRNA的表达水平。 结果：在镁锌合金表面MC3T3-E1细胞黏附性能优于左旋聚乳酸表面;镁锌合金表面培养的MC3T3-E1细胞在实验期间能持续表达整合素β1mRNA,表达水平随着时间延长而增高(P < 0.01),但与左旋聚乳酸相比差异无显著性意义(P > 0.05)。 结论：镁锌合金表面MC3T3-E1细胞黏附性能优于左旋聚乳酸表面,但并非通过诱导整合素β1的表达水平来介导的。 关键词：镁锌合金;成骨细胞;可吸收生物材料;整合素;黏附     

miR-429对低氧诱导的缺血性脑卒中的血管生成作用机制研究

目的探讨mi R-429对低氧诱导的缺血性脑卒中的血管生成作用机制研究。方法线栓法建立永久性局灶脑缺血大鼠模型,随机分为4组进行不同处理:(1)antagomi R-Ctr组:常氧预处理并注射antagomi R-Ctr;(2)antagomi R-429组:常氧预处理并注射antagomi R-429;(3)Co Cl2+antagomi R-Ctr组:Co Cl2预处理并注射antagomi R-Ctr;(4)Co Cl2+antagomi R-429组:Co Cl2预处理并注射antagomi R-429。Co Cl2溶液和antagomi R-Ctr/429的剂量分别是30mg·kg-1和12mg·kg-1。通过实时荧光定量核酸扩增检测系统(QPCR)和蛋白印迹试验(Western blot)检测各组HIF-1α、VEGF和mi R-429的表达,通过异硫氢酸荧光素葡聚糖(FITC-Dextran)和氯化三苯基四氮唑(TTC)染色分别检测微血管密度和梗死体积。结果在脑缺血后第7d和21d,Co Cl2预处理和antagomi R-429能诱导大鼠低氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)的表达,降低mi R-429的表达,并促进血管生成和缩小梗死体积;脑缺血后第7d,Co Cl2+antagomi R-429组较其余3组HIF-1α和VEGF的表达最高,mi R-429的表达最低,微血管密度最大且梗死体积最小,且在脑缺血后第21d,此趋势更明显。结论 Antagomi R-429促进低氧诱导的脑缺血大鼠的血管生成,mi R-429可作为治疗缺血性脑卒中的新靶点。     

Comparative effects of metal chelating agents on the neuronal cytotoxicity induced by copper (Cu+2), iron (Fe+3) and zinc in the hippocampus

The ability of metal chelating agents to prevent neuronal death caused by intra-hippocampal injections of cupric sulphate, ferric citrate and zinc chloride was investigated. Ammonium tetrathiomolybdate was itself toxic after injection into the hippocampus, but this toxicity was reduced by formation of a metal ion/tetrathiomolybdate complex with Cu+2. Disodium bathocuproine disulphonate (BCDS) prevented neuronal death caused by Cu+2, but not that induced by Fe+3 or Zn+2. Desferrioxamine prevented death caused by Fe+3, had no significant effect of the toxicity of Zn+2, and increased that caused by Cu+2. Even though N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) has a higher affinity for Cu+2 than for Zn+2, TPEN had no effect on the toxicity of Cu+2 while totally preventing damage caused by Fe+3 or Zn+2. Ethylenediaminetetra-acetic acid (EDTA) prevented the toxicity of all three metal ions. Motor seizure activity occurred in most rats after injections of Fe+3; or combinations of Cu+2 plus TPEN, or 4 nmol Fe+3 plus 0.1 nmol desferrioxamine. However, apart from the low dose desferrioxamine/Fe+3 combination, only the occasional brain contained seizure-induced neuronal loss in limbic regions outside the injected hippocampus, and these brains were not used for analysis. Seizure activity was found even with very low levels of Cu+2 with a fixed amount of TPEN (a ratio of Cu+2/TPEN of 1:100), but the extent of hippocampal damage in these brains was not significantly different to that caused by injections of saline. These studies demonstrate that idiosyncratic interactions can occur between metal ions and chelating agents. Thus further investigations are needed before chelating agents can be examined for their protective properties in various neurodegenerative diseases.     

Cations differentially affect subpopulations of L-glutamate receptors in rat synaptic plasma membranes

Several cations were examined for their ability to specifically affect one of the 3 L-glutamate (L-Glu) binding sites in rat forebrain synaptic plasma membranes (i.e. Na+-dependent, Cl--dependent and Cl--independent). Na+-dependent binding was potently inhibited by K+ and NH4+ ions. Other monovalent cations tested (Cs+, Li+, triethylammonium) had no effect on this binding site. Polyvalent cations (Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Cr3+) also had little effect on the Na+-dependent L-Glu binding site. Cl--dependent L-Glu binding was potently inhibited by Na+ ions but was not affected by other monovalent ions. All of the divalent cations were potent inhibitors of both Cl--dependent and -independent binding. The results show that these binding sites of L-Glu can be distinguished by their response to cations and suggest possible novel modes of regulation in vivo.     

Protection against amyloid beta peptide toxicity by zinc

Zinc (Zn) is an essential element in normal development and biology, although it is toxic at high concentrations. Recent studies show that Zn at high concentrations accelerates aggregation of amyloid beta peptide (Abeta), the major component of senile plaques in Alzheimer's disease (AD). This study reports the effect of varying Zn concentrations on Abeta toxicity and the mechanism by which low concentrations function in a protective role. At Abeta/Zn molar ratios of 1:0.1 and 1:0.01, Zn produces significant protection against Abeta toxicity in cultured primary hippocampal neurons. At higher concentrations (1:1 molar ratio), Zn offers no protection or enhances Abeta toxicity. The protective effect of Zn against Abeta toxicity is due in part to the enhancement of Na+/K+ ATPase activity which prevents the disruption of calcium homeostasis and cell death associated with Abeta toxicity. Analysis of Na+/K+ ATPase activity in cultured rat cortical cells indicated that Zn exposure alone afforded a 20% increase in enzyme activity, although the differences were statistically insignificant. However, in cortical cultures exposed to a toxic dose of Abeta (50 microM), Zn at concentrations of 5 and 0.5 microM led to significant increases in Na+/K+ ATPase activity compared with levels in cells treated with Abeta alone. Zn at a 1:1 molar ratio (50 microM) led to a significant decrease in enzyme activity. Together, these data suggest that Zn functions as a double-edged sword, affording protection against Abeta at low concentrations and enhancing toxicity at high concentrations.     

Lithium ion "cyclotron resonance" magnetic fields decrease seizure onset times in lithium-pilocarpine seized rats

The cyclotron resonance equation predicts that the frequency of an applied magnetic field that might optimally interact with a single ion species may be computed as a function of the charge-to-mass ratio of the ion and the strength of the background static magnetic field. The present study was undertaken to discern the applicability of this equation for optimizing lithium ion utilization in the rat, as inferred by the predicted magnetic "ion resonance "field-induced shift of lithium's dose-dependent curve for seizure onset times (SOTs) when combined with the cholinergic agent pilocarpine. Groups of rats were administered 1.5 thru 3 mEq/kg lithium chloride (in 0.5 mEq/kg increments) and exposed to reference conditions or to one of three intensities (70 nanoTesla, 0.8 microTesla, or 25 microTesla) of a 85 Hz magnetic field calculated to resonate with lithium ions given the background static geomagnetic field of approximately 38,000 nanoTesla (0.38 Gauss). A statistically significant quadratic relationship for SOT as a function of magnetic field intensity (irrespective of lithium dose) was noted: this U-shaped function was characterized by equal SOTs for the reference and 25 microTesla groups, with a trend toward shorter SOTs for the 70 nanoTesla and 0.8 microTesla groups. Although not predicted by the equations, this report extends other findings suggestive of discrete intensity windows for which magnetic field frequencies derived from the cyclotron ion resonance equation may affect ion activity.     

Metal chelates of L-DOPA for improved replenishment of dopaminergic pools.

An exploratory study consisting of physiochemical and animal experiments was undertaken with the objective of developing one or more metal-L-DOPA chelate systems for an improved transport of L-DOPA into the brain. This approach is based on a theoretical speculation that the pyridoxal-dependent decarboxylation of L-DOPA in the precerebral areas might be obviated by an appropriate metal chelation of the aminocarboxylate end of the L-DOPA molecule. Equilibium studies on the interactions of L-DOPE with Cu2+,Zn2+, Co2+, Mg2+ and Fe2+ ions and their ATP chelates were carried out in order to examine the conditions for the selective binding of the terminal amine group. Metal chelate systems for in vivo transport experiments were selected viz., Cu2+ or Zn2+-L-DOPA (1:2) and Cu2+ or Zn2+-ATP-L-DOPA (1:1:1) which contained the amine-bound metal ion in a completely coordinated form. Results of in vivo studies involving the intraperitoneal administration of 14C-and 3H-LABELED L-DOPA compounds have shown a 100-150% increase in the transport of L-DOPA into the brain by using the Cu2+ and Zn2+ chelates over that effected by using the unchelated L-DOPA. A chromatographic analysis of the brain homogenates showed that only 6% of the overall radioactivity of the brain could be attributed to 3-methoxytyrosine, and the remaining activity was due to DOPA, dopamine and norepinephrine. The transport effectiveness was also compared with that obtained by using the combination drug, RO4-4602 + L-DOPA.     

Zinc ions are endogenous modulators of neurotransmitter-stimulated capacitative Ca2+ entry in both cultured and in situ mouse astrocytes

Astrocytes express a variety of metabotropic receptors and their activation leads to a biphasic Ca2+ response due to Ca2+ release from intracellular stores and subsequent capacitative Ca2+ entry. We performed Ca2+ imaging with Fura-2 on cultured mouse astrocytes and showed that extracellular zinc reversibly blocks the capacitative Ca2+ entry following application of the metabotropic ligands ATP, glutamate and endothelin-1. Zinc blocked the plateau phase of the ligand-triggered Ca2+ responses. When ligands were repetitively applied in the presence of zinc the calcium responses progressively decayed and even disappeared, indicating that capacitative Ca2+ entry is required to refill the stores. Zinc inhibited the capacitative Ca2+ entry with a K(i) of approximately 6 microM, which is well within the physiological concentration range of zinc found in the brain. Application of the reducing agent DTT prevented the blocking effect by zinc ions but not the inhibition elicited by the nonphysiological metal ions Gd3+ and La3+, indicating that zinc has a distinct binding site. To monitor the capacitative Ca2+ entry in astrocytes in situ and to determine the effect of zinc on this pathway we utilized X-rhod-1 imaging in hippocampal slices of a transgenic mouse line with green fluorescent astrocytes. Zinc affected the repetitive metabotropic Ca2+ response in the following fashion: (i) after depleting stores in Ca(2+)-free solution, re-addition of Ca2+ led to an influx of Ca2+ via a zinc-sensitive Ca2+ entry route; (ii) with repetitive application of metabotropic ligands, Ca2+ responses became smaller and even disappeared in the presence of zinc. We conclude that zinc, which is co-released from glutamatergic synaptic vesicles upon neuronal activity, has a major impact on shaping the astrocytic calcium responses.     

Differential effects of transition metal cations on the conformation and biological activities of nerve growth factor

Direct effects of Zn²⁺ on the conformation and biological activity of nerve growth factor (NGF) have previously been described. Zn²⁺ binds to specific coordination sites within NGF and induces conformational changes within domains that participate in receptor recognition processes. Recent theoretical considerations indicate that other transition metal cations (particularly, Cu²⁺and Pd²⁺) are capable of forming similar complexes with NGF. Inactivation of NGF by transition metal cations is inhibitory to neuronal regeneration and sprouting, and can lead to cell death under conditions where NGF is required for survival in PC12 cells. In this study we investigated the influence of various metal ions on NGF conformation, geometry of NGF amino terminal peptide and NGF-mediated biological effects in FC12 cells. A number of metal ions (Zn²⁺, Cu²⁺ and Pd²⁺) alter NGF conformation in cell-free assays and inhibit NGF-mediated cell survival. Other metals have been shown to be either toxic to PC12 cells by mechanisms independent of NGF activity (e.g. Ag⁺, Hg²⁺) or non-toxic to the cells under conditions tested (e.g. Al³⁺, Cr³⁺). In conclusion, several metal cations are capable of inhibiting NGF activity, thereby blocking NGF-mediated cell survival and plasticity.