力学刺激对软骨细胞细胞骨架的影响

目的 探讨不同应力刺激下软骨细胞细胞骨架的荧光强度和形态分布的变化。方法 取新西兰兔关节软骨细胞，分离传代增殖然后将第3代软骨细胞分别给以3组不同的动态（1Hz，500με 1000με 1500με）和静态（0Hz，500με 1000με 1500με）力学刺激分组培养，然后行细胞骨架肌动蛋白（actin）、中间丝波形蛋白（vimentin）、微管（tubulin）的免疫荧光染色，吸光法对标本进行测定，分析荧光强度和细胞骨架分布的变化。结果 软骨细胞在未受到应力刺激时其分布相对分散并显示比较均匀，在力学刺激后，细胞骨架发生的排列和强度均发生了变化。vimentin在动态的力学刺激下荧光强度逐渐增强，Actin先减弱后逐步增强，而Tublin是先增强在减弱再增强。vimentin、actin和tublin的荧光强度在静态的力学刺激下有明显的一致性的变化，随压力的增加其强度明显的下降，其排列疏松变化明显。结论 在力学刺激下，细胞骨架发生的明显的变化，而在各种变化中，其变化和力学刺激的大小和时间和刺激的方式有关系。     

小鼠舌肌发育相关基因的功能聚类分析

目的：研究小鼠舌肌发育的分子调控机制。方法：取胚胎第13．25天（E13．25）及 E15．5小鼠舌组织。应用 Affy-metrix Mouse GeneChip，对胎鼠舌发育过程中的差异基因进行筛选。应用 DAVID 网络分析工具对基因进行功能和聚类分析。结果：基因功能和聚类分析表明，在 E13．25高表达的基因主要与细胞周期相关因子（Exo1、Gsk3B、Kif20b、Skp2）和细胞粘附因子（Neo1、lama1）等相关。在 E15．5高表达的基因主要与细胞骨架（titin、Hspb7）相关。结论：小鼠舌组织增殖和特化与细胞周期和细胞粘附基因相关，舌组织分化和成熟主要与细胞骨架相关。     

热打击对人骨骼肌细胞通透性、细胞骨架及细胞周期的影响

目的 研究热打击对培养人骨骼肌细胞(HSKMC)通透性、细胞骨架及细胞周期影响.方法 流式细胞仪钙离子内流检测热打击对HSKMC细胞膜通透性的影响,考马斯亮蓝R-250染色法检测热打击对HSKMC细胞骨架影响,流式细胞仪检测热打击对HSKMC细胞周期改变.结果 给予培养人HSKMC细胞不同温度梯度热打击1h后,43℃热打击组钙离子流式检测的中位数为91.63,37℃对照组中位数为22.98.同对照组相比,随着热打击程度加强,显微镜高倍镜下可见骨骼肌细胞骨架逐渐出现变粗、变短、出现明显的应力纤维.骨骼肌细胞在热打击情况下,各组G0/G1期DNA含量在44.13～62.98之间,与正常对照组对比,当细胞离开热打击环境后培养18 h前G0/G1期DNA含量明显高于对照组,培养第18小时恢复才同对照组基本相同.结论 热打击的情况下造成细胞钙离子内流效应,导致细胞内钙超载.可改变HSKMC骨架结构,使骨架失去正常的网状有序排列结构,间隙增大.细胞周期出现阻滞,细胞被阻滞在G0/G1期.     

Cytoskeletal disruption is the key factor that triggers apoptosis in okadaic acid-treated neuroblastoma cells

Okadaic acid (OA) is a tumour promoter that induces apoptosis in several cell models. Following previous findings, the objective of this work was to elucidate the pathways involved in OA-triggered apoptosis in BE(2)-M17 cells by using a combination of pharmacological agents and apoptosis-related assays. OA-induced apoptosis involves disruption of F-actin cytoskeleton, activation of caspase-3, collapse of mitochondrial membrane potential, DNA fragmentation and decreased levels of monomeric Bcl-2 and Bax proteins. All the agents tested were unable to obliterate changes in F-actin levels, caspase-3 activation or DNA fragmentation, but all of them prevented OA-induced decrease of mitochondrial potential and changes in Bax/Bcl-2 levels. Taken together, these results demonstrate that collapse of mitochondrial membrane potential is accessory in the execution of apoptosis, which is directly dependent on cytoskeletal changes. Mitochondrial changes are mediated by complex associations among the Bcl-2 proteins. Cytochrome c release from mitochondria is a late event, occurring 24 h after OA exposure. Moreover, okadaic acid triggers activation of upstream caspases resembling the extrinsic pathway of apoptosis.     

静态拉伸应变对人牙周膜成纤维细胞的影响

目的：研究人牙周膜成纤维细胞(PDLF)在机械拉伸应变作用下，细胞和细胞核投影面积及其细胞骨架的改变情况，探讨PDLF的形态、功能和应变之间的关系。方法：采用自制的细胞体外静态机械加载装置用不同的拉伸应变量加力于细胞上，通过激光扫描共聚焦显微镜观察细胞骨架的形态及其改变情况，并进行统计分析。结果：在8％、12％、16％力值组，细胞和细胞核投影面积随着加力时间和力值的增加而每天递增，肌动蛋白纤维也随之逐渐增粗，排列更有规律；而在20％力值组，其结果则反之。结论：静态拉伸应变可影响牙周膜成纤维细胞的骨架。     

烧伤血清刺激对大鼠肠上皮细胞结构和粘弹性的影响

目的　动态观察烧伤血清对体外肠上皮细胞 (IEC)骨架和细胞生物力学 (粘弹性 )的影响。　方法　培养大鼠肠上皮细胞株IEC 6 ,用烧伤血清刺激后 ,通过细胞骨架免疫组化、细胞ELISA法定量分析以及粘弹性测定技术 ,动态观察IEC致伤前后的变化。　结果　IEC在烧伤血清作用早期 ,骨架蛋白的表达即明显降低 ,微丝、微管蛋白阳性信号减弱 ,细胞粘弹性下降。　结论　细胞骨架的损伤可引起细胞脆性增加、粘弹性下降 ,导致细胞生物力学特性的改变。这种变化 ,可能直接参与烧伤后肠上皮细胞损伤的发生。     

内皮细胞白细胞黏附分子-1对猪眼小梁细胞形态和细胞骨架的作用

目的研究内皮细胞白细胞黏附分子-1(ELAM-1)对猪眼小梁细胞形态和肌动蛋白细胞骨架的作用，探讨青光眼的发病机制。方法原代培养的猪眼小梁细胞，经白细胞介素1(IL-1)刺激活化后，结合ELAM-1抗体或IgG进一步交联，观察细胞形态、细胞间连接的动态改变及肌动蛋白和细胞厚度的改变。结果未经IL-1作用的猪眼小梁细胞不表达ELAM-1，IL-1作用后猪眼小梁细胞表达ELAM-1。经过IL-1活化的猪眼小梁细胞，结合ELAM-1抗体或IgG进一步交联后，细胞间隙扩大，细胞变圆、变厚，细胞立体感增强；肌动蛋白变稀疏，荧光弥散，细胞的刚性降低，细胞厚度增加。结论猪眼小梁细胞能表达ELAM-1，表达的ELAM-1通过与抗体结合或同时通过IgG交联影响肌动蛋白细胞骨架，改变细胞形态。     

A comparative study of the effect of oxidative stress on the cytoskeleton in human cortical neurons

Cytoskeleton disruption is a process by which oxidative stress disrupts cellular function. This study compares and contrasts the effect of oxidative stress on the three major cytoskeleton filaments, microfilaments (MFs), microtubule (MT), and vimentin in human cortical neuronal cell line (HCN2). HCN2 cells were treated with 100 microM tertiary butylhydroperoxide (t-BuOOH), a free radical generating neurotoxin for 1, 3, or 6 h. Cell viability studies demonstrated significant cell death although the morphology studies showed that there was a substantial loss in neurites of neurons treated with t-BuOOH for 6 h. Because the cytoskeleton plays a role in neurite outgrowth, the effect of oxidative stress on the cytoskeletal was studied. In neurons subjected to oxidative stress for 30 min or 1 h, there were no major changes in microfilament distribution though there was altered distribution of microtubule and vimentin filaments as compared to controls. However, loss and disruption of all the three cytoskeletal filaments was observed at later times (3 and 6 h), which was confirmed by Western Blot analysis. Further studies were done to measure the gene expression levels of actin, tubulin, and vimentin. Results indicated that the overall loss of the cytoskeletal proteins in neurons treated with free radical generating toxin might not be a direct result of the downregulation of the cytoskeletal genes. This study shows that free radical generation in human neurons leads to the disruption of the cytoskeleton, though there may be a difference in the susceptibility to oxidative stress among the individual components of the cytoskeletal filaments.     

Experimental studies of membrane tethers formed from human neutrophils

Membrane tethers (thin, cylindrical pieces of membrane) have been implicated in the rolling of neutrophils along the endothelium. In our studies, these tethers were formed from passive, stimulated (0.1 M fMLP), and osmotically swollen (170–180 mOsm) human neutrophils; as well as neutrophils treated with 0.3 M latrunculin A to disrupt the cytoskeleton. This tether formation was accomplished by micropipette suction of latex beads coated with antibodies to proteins on the neutrophil membrane surface. From plots of force versus velocity for the tether formation process, we calculated adhesion energies per unit area of the lipid membrane to the cytoskeleton and the viscous resistance (effective viscosity) that occurs during the formation of these tethers at finite velocity. Most of the properties of the neutrophil were altered once it had been treated as described above. We were also able to show mechanical reversibility of membrane tethers, as well as an unexpected formation rate at high tether forces. Since membrane tethers have been implicated in the rolling of neutrophils, then the changes in tether formation may ultimately alter how these cells roll. © 2002 Biomedical Engineering Society. PAC2002: 8716Dg     

Microtubules in cardiac toxicity and disease

Microtubules (MTs) are dynamic, cytoskeletal fibers that are found in every eukaryotic cell type. MTs serve a wide range of functions, including cell division, membrane and vesicle transport, and motility. As such, MTs play pivotal roles in cardiac development and function. Agents that disrupt normal MT function, including such therapeutic agents as vincristine and paclitaxel, have also been shown to affect essential cardiac activities such as sarcomere mechanics, beat rate, and the secretion of important molecules (e.g., atrial natriuretic factor). Disease states that lead to either ischemia- or pressureoverload-induced cardiac hypertrophy also alter the microtubule cytoskeleton in several ways. A fuller understanding of the contributions of MTs to cardiac development and function will be necessary to minimize the deleterious, side effects of the therapeutic application of MT-disrupting drugs. This review summarizes current hypotheses and experimental results that demonstrate the central role of MTs in heart cell function and disease.