秋水仙素和长春花碱对肝癌细胞粘弹性的影响

采用微管吸吮技术测定了正常肝细胞和肝癌细胞的粘弹特性,以三元素标准线性固体模型拟合实验结果,进一步研究秋水仙素和长春花碱处理后肝细胞和肝癌细胞粘弹性系数的变化。结果表明,肝癌细胞的弹性系数较之肝细胞的相应值增高,在秋水仙素和长春花碱作用下肝细胞和肝癌细胞的粘弹特性呈现不同的效应方式和强度。上述结果可能反映了两种细胞微管结构和机能状态的差异,癌细胞粘弹性的改变可能影响到浸润和转移特性以及癌细胞与其微观力学环境的相互作用。     

肝癌细胞与胶原蛋白Ⅰ裱衬表面粘附特性

本文以正常胎肝细胞的原代培养及肝癌细胞的传代培养为基础,应用微管吸吮技术在单个细胞水平上研究肝实质细胞癌细胞在胶原蛋白Ⅰ裱衬表面的粘附力学特性。实验将正常原代肝细胞与肝癌细胞进行对照,阐明肝癌细胞与胶原蛋白Ⅰ裱衬表面粘附行为的时间与浓度依赖性,结果表明肝癌细胞与胶原蛋白Ⅰ裱衬表面具有更强的粘附力。这一研究对肝癌转移的定量研究有方法学的参考意义。     

细胞松弛素D、秋水仙素作用下大鼠肝癌细胞中黏弹性的研究

采用微管吮吸技术测定大鼠肝癌细胞的黏弹性;研究了秋水仙素、细胞松弛素D以及两者混合作用后对于肝癌细胞黏弹性的影响。结果表明:用CD处理癌细胞后发现癌细胞的弹性系数K1明显下降。与对照组相比:微丝骨架被CD抑制后,在加入Col后肝癌细胞的弹性系数K1显著降低;而微管骨架被col抑制后,在加入CD后肝癌细胞的弹性系数K1、K2和μ无明显变化。提示在微管骨架系统完整的情况下,微丝对肝癌细胞的黏弹性系数的的影响起主要作用。而微管骨架系统受到破坏后,微丝需借助于微管网络的作用来影响细胞的黏弹性。本研究对揭示癌细胞中骨架系统之间的交互作用对于细胞黏弹性的影响提高实验依据。     

肝癌细胞整合素受-配体比与其粘附稳定性的相关性实验研究

整合素与其胞外基质配体间的相互作用对调节细胞的粘附和运动起着重要的作用.肝癌细胞的胞外基质减少,而整合素β1的表达却增高,其比例失衡影响肝癌细胞的粘附与运动行为.作者通过细胞形态学观察、图像分析,微管吸吮和流式细胞仪等手段,对肝癌细胞、正常肝细胞的整合素表达水平、裱衬Fn前后肝癌细胞的运动能力及粘附力进行检测和定量分析,发现肝癌细胞的整合素表达量高于正常肝细胞;肝癌细胞粘附力较正常肝细胞低,迁移速度增快,补充适当浓度胞外配体Fn可使胞外受配体比例恢复到正常肝细胞的整合素表达水平,裱衬Fn后肝癌细胞的粘附力增强,细胞运动能力减弱.这些结果说明胞外配体Fn对肝癌细胞整合素表达有下调作用,肝癌细胞的受.配体比例是影响其粘附和运动的因素之一.     

微管吸吮和半无限体模型在鼠成骨细胞粘弹性研究中的应用

利用微管吸吮技术研究了体外培养幼鼠颅盖骨成骨细胞的被动变形特性,测量了其在阶跃负压下吸入长度随时间的变化过程,并用标准线性粘弹性半无限体模型研究了成骨细胞的粘弹性特性;并研究了秋水仙素对成骨细胞弹性性质的影响,经秋水仙素处理后的成骨细胞与正常组成骨细胞相比,其杨氏模量没有显著性改变。本研究工作为进一步研究成骨细胞的离体及在体的主动变形机制提供了基础数据。     

衰老标记蛋白30 mRNA在癌细胞中的表达研究

目的:检测衰老标记蛋白(SMP)30 mRNA在不同癌细胞系中的表达情况,探讨其在不同细胞中的表达差异。方法:分别采用RT-PCR与荧光定量PCR检测SMP30 mRNA在正常肝细胞、肝癌细胞、胃癌细胞、乳腺癌细胞、宫颈癌细胞中的表达,并用SPSS13.0进行统计学分析。结果:SMP30 mRNA在所有被检测的细胞株中均有表达,在癌细胞中的相对表达量分别为肝癌细胞(0.926±0.340)、胃癌细胞(0.922±0.379)、乳腺癌细胞(0.614±0.356)、宫颈癌细胞(0.608±0.346),而在正常肝细胞中为0.175±0.158,显示SMP30 mRNA在癌细胞中的表达量较正常肝细胞中高(P0.05),且在肝癌细胞中的表达量比在其他癌细胞中更高。结论:SMP30 mRNA在癌细胞中的表达高于正常肝细胞,且在肝癌细胞中的表达高于其他癌细胞,具有临床应用价值。     

分泌人体肝癌单克隆抗体的两个小鼠杂交瘤株及其抗体的专一性分析(简报)

有证据表明人体肝癌细胞表面膜抗原的成份与正常肝细胞的有差异。应用125I UdR释放试验也证明肝癌患者的周围血淋巴细胞对体外培养的肝癌细胞有细胞毒作用。表明患者的免疫系统有可能识别这些膜抗原的差异性。但除了与人体肝癌有交义反应的胚胎肝抗原的性质有些初步报道以外,人体肝癌相关抗原的种类     

天花粉蛋白对滋养层细胞专一损伤机制的研究

在体外培养条件下,天花粉蛋白胶体金以受体介导的内吞方式进入滋养层细胞和绒癌细胞,最终进入胞质溶胶着在核糖体上,经相同处理的肝癌细胞,绿猴肾细胞和正常鼠胚肝细胞,金颗粒既不与这些细胞表面结合,也没有被专一内吞的现象。分别用ＢＳＡ,转铁蛋白活化的胶体金处理滋养层细胞和绒癌细胞的方式与天花粉蛋白的结果相同,先以肝癌单抗处理也不影响天花粉蛋白的结果相同,先以肝癌单抗处理也不影响天花粉蛋白的肝癌单抗结合物进     

人胎肝细胞表面膜抗原的提取

近年来,我们取四个月左右胎肝细胞制备的异种抗血清,经成年人组织细胞吸收后,用于离体培养的人体肝癌细胞的免疫膜荧光和铁蛋白标记抗体实验,发现这类细胞除能合成甲胎蛋白外,其细胞表面还有着与胎肝细胞和未成熟的造血细胞表面有交叉免疫反应的膜抗原。提取和分     

integrin β1在不同细胞周期肝癌细胞与内皮细胞粘附中的作用

探讨了integrin β1在不同细胞周期人肝癌细胞(SMMC-7721)上的表达和在肝癌细胞与人脐静脉内皮细胞粘附过程中的作用.未同步处理的肝癌细胞(对照组)各细胞周期时相百分比为G0/G1期53.51%、G2/M期11.01%、S期35.48%,采用胸腺嘧啶脱氧核苷、秋水仙碱顺序阻断和胸腺嘧啶脱氧核苷双阻断后释放培养的方法获得G1期和S期的肝癌细胞,其同步率分别为74.09%和98.29%.G1期肝癌细胞integrin β1表达的荧光强度较S期和对照组相应值明显降低.利用微管吸吮技术定量研究了肝癌细胞与内皮细胞之间的粘附力学特性,发现G1期肝癌细胞的粘附力值比S期相应值明显降低(P＜0.01),而S期的粘附力值与对照组比较无明显差别,integrin β1在肝癌细胞与内皮细胞粘附过程中的贡献约50%.结果提示胸腺嘧啶脱氧核苷和秋水仙碱能较好地将肝癌细胞同步于G1期和S期,integrin β1在SMMC-7721肝癌细胞上的表达水平呈现周期差异,在肝癌细胞与内皮细胞粘附过程中,S期细胞可能起的作用更大,integrin β1在这一粘附过程中起着重要作用.     

Comparison of the viscoelastic properties of normal hepatocytes and hepatocellular carcinoma cells under cytoskeletal perturbation

The viscoelastic properties of both hepatocytes and hepatocellular carcinoma (HCC) cells were measured by means of a micropipette aspiration technique. Experimental results were analyzed with a three-element standard linear solid model, in which an elastic element, K1, is in parallel with a Maxwell element composed of another elastic element, K2, in series with a viscous element, mu. Further, we investigated the relevance of viscoelastic properties of these two types of cells to the cytoskeleton structures by treating cells with three cytoskeletal perturbing agents, namely cytochalasin D (CD), colchicine (Col) and vinblastine (VBL). The results showed that the elastic coefficients, but not viscous coefficient of HCC cells (K1 = 103.6 +/- 12.6 N m-2, K2 = 42.5 +/- 10.4 N m-2, mu = 4.5 +/- 1.9 Pa s, n = 30), were significantly higher than the corresponding values for hepatocytes (K1 = 87.5 +/- 12.1 N m-2, K2 = 33.3 +/- 10.3 N m-2, mu = 5.9 +/- 3.0 Pa s, n = 24). Upon treatment with CD, the viscoelastic coefficients of both hepatocytes and HCC cells decreased uniformly, with magnitudes for the decrease in elastic coefficients of HCC cells (K1: 68.7 to 81.7 N m-2, 66.3 to 78.9%; K2: 34.5 to 37.1 N m-2, 81.2 to 87.3%) larger than those for normal hepatocytes (K1: 42.6 to 49.8 N m-2, 48.7 to 56.9%; K2: 17.2 to 20.4 N m-2, 51.7 to 61.3%). There was a smaller decrease in the viscous coefficient of HCC cells (2.0 to 3.4 Pa s, 44.4 to 75.6%) than that for hepatocytes (3.0 to 3.9 Pa s, 50.8 to 66.1%). Upon treatment with Col and VBL, the elastic coefficients of hepatocytes generally increased or tended to increase while those of HCC cells decreased. The differences in either the pattern or the magnitude of the effect of cytoskeletal perturbing agent on the viscoelastic properties between HCC cells and hepatocytes might possibly reflect differences in the state of the cytoskeleton structure and function, or in the cells' sensitivity to perturbing agent treatment between these two types of cells. Changes in the viscoelastic properties of cancer cells might well affect tumor cell invasion and metastasis as well as interactions between tumor cells and their micro-mechanical environments.     

Determination of the Poisson's ratio of the cell: recovery properties of chondrocytes after release from complete micropipette aspiration

Chondrocytes in articular cartilage are regularly subjected to compression and recovery due to dynamic loading of the joint. Previous studies have investigated the elastic and viscoelastic properties of chondrocytes using micropipette aspiration techniques, but in order to calculate cell properties, these studies have generally assumed that cells are incompressible with a Poisson's ratio of 0.5. The goal of this study was to measure the Poisson's ratio and recovery properties of the chondrocyte by combining theoretical modeling with experimental measures of complete cellular aspiration and release from a micropipette. Chondrocytes isolated from non-osteoarthritic and osteoarthritic cartilage were fully aspirated into a micropipette and allowed to reach mechanical equilibrium. Cells were then extruded from the micropipette and cell volume and morphology were measured throughout the experiment. This experimental procedure was simulated with finite element analysis, modeling the chondrocyte as either a compressible two-mode viscoelastic solid, or as a biphasic viscoelastic material. By fitting the experimental data to the theoretically predicted cell response, the Poisson's ratio and the viscoelastic recovery properties of the cell were determined. The Poisson's ratio of chondrocytes was found to be 0.38 for non-osteoarthritic cartilage and 0.36 for osteoarthritic chondrocytes (no significant difference). Osteoarthritic chondrocytes showed an increased recovery time following full aspiration. In contrast to previous assumptions, these findings suggest that chondrocytes are compressible, consistent with previous studies showing cell volume changes with compression of the extracellular matrix.     

Hepatocellular Carcinoma Cells Deteriorate the Biophysical Properties of Dendritic Cells

Dendritic cells (DCs) are potent antigen-presenting cells and induce antigen-specific immune responses in the organism. The dysfunction of DCs has been implicated in tumor-bearing host. In order to elucidate the effects of tumor microenvironment on the functions of DCs from interdisciplinary aspects, we characterized the biophysical properties of DCs co-cultured with hepatocellular carcinoma cells (HCC). The results showed that the biophysical characteristics of immature and mature DCs were severely impaired by HCC compared with those under normal conditions, including the increased osmotic fragilities, decreased cell membrane fluidities, increased membrane viscoelastic properties, dysfunction and increased expression of cytoskeleton protein F-actin, as well as the deteriorated transendothelium migration. The impaired biophysical properties of DCs may be one of many aspects of the immune escape mechanisms of tumors. These results are clinically and instructionally significant with regard to how to enhance efficiency of the anti-tumor therapy based on DCs. Zhu Zeng and Weijuan Yao contributed equally to this work.     

Microporous cellulosic scaffold as a spheroid culture system modulates chemotherapeutic responses and stemness in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) treatments are evaluated by two-dimensional (2D) in vitro culture systems, despite their limited ability to predict drug efficacy. The three-dimensional (3D) microporous scaffold provides the possibility of generating more reliable preclinical models to increase the efficacy of cancer treatments. The physical properties of a microporous cellulosic scaffold were evaluated. The cellulosic scaffold was biocompatible and had a highly porous network with appropriate pore size, swelling rate, and stiffness of cancer cell cultures. Cellulosic scaffolds were compared with 2D polystyrene for the culture of HepG2 and Huh7 human HCC cells. Cellulosic scaffolds promoted tumor spheroid formation. Cells cultured on scaffolds were more resistant to chemotherapy drugs and showed upregulation of EpCAM and Oct4. The migration ability of HCC cells cultured on scaffolds was significantly greater than that of cells grown in 2D cultures as evidenced by the downregulation of E-cadherin. In addition, the proportion of CD44+/CD133+ HCC cancer stem cells (CSCs) was significantly greater in cells cultured on scaffolds than in those grown in 2D cultures. These findings suggest that cellulosic scaffolds effectively mimic the in vivo tumor behavior and may serve as a platform for the study of anticancer therapeutics and liver CSCs.     

Mechanosensitivity of a Rapid Bioluminescence Reporter System Assessed by Atomic Force Microscopy

Cells are sophisticated integrators of mechanical stimuli that lead to physiological, biochemical, and genetic responses. The bioluminescence of dinoflagellates, alveolate protists that use light emission for predator defense, serves as a rapid noninvasive whole-cell reporter of mechanosensitivity. In this study, we used atomic force microscopy (AFM) to explore the relationship between cell mechanical properties and mechanosensitivity in live cells of the dinoflagellate Pyrocystis lunula. Cell stiffness was 0.56 MPa, consistent with cells possessing a cell wall. Cell response depended on both the magnitude and velocity of the applied force. At the maximum stimulation velocity of 390 μm s⁻¹, the threshold response occurred at a force of 7.2 μN, resulting in a contact time of 6.1 ms and indentation of 2.1 μm. Cells did not respond to a low stimulation velocity of 20 μm s⁻¹, indicating a velocity dependent response that, based on stress relaxation experiments, was explained by the cell viscoelastic properties. This study demonstrates the use of AFM to study mechanosensitivity in a cell system that responds at fast timescales, and provides insights into how viscoelastic properties affect mechanosensitivity. It also provides a comparison with previous studies using hydrodynamic stimulation, showing the discrepancy in cell response between direct compressive forces using AFM and those within flow fields based on average flow properties.     

Fluvastatin, a lipophilic statin, induces apoptosis in human hepatocellular carcinoma cells through mitochondria-operated pathway

Fluvastatin, a lipophilic statin, was known to inhibit proliferation and induce apoptosis in many cancer cells. Its potential anticancer was evaluated in three hepatocellular carcinoma (HCC) cell lines (HepG2, SMMC-7721 and MHCC-97H). Cells were treated with fluvastatin in vitro and its effect on cell proliferation, cell cycle, invasion and apoptosis was determined. Mechanism of apoptosis induced by fluvastatin on HCC cell lines was also investigated through western blotting and mitochondrial membrane potential (MMP) analysis. It was observed that fluvastatin inhibited proliferation of HCC cells by inducing apoptosis and G2/M phase arrest in a dose-dependent manner. The results of cell invasion assay revealed that fluvastatin significantly decreased the invasion potency of HCC cells. A mitochondria-operated mechanism for fluvastatin induced apoptosis might be involved and was supported by Western blotting and MMP analysis. After fluvastatin treatment, expression of Bcl-2 and procaspase-9 were downregulated, cytochrome c (cytosolic extract), Bax and cleaved-caspase-3 protein expression were increased. Furthermore, a breakdown of MMP in HCC cells was observed. To conclude, these results have provided a rationale for clinical investigations of fluvastatin in future as a potential anticancer reagent for growth control of HCC.     

Effects of gibberellin and colchicine on microfibril arrangement in epidermal cell walls of Vigna angularis Ohwi et Ohashi epicotyls

Gibberellic-acid (GA₃) treatment of azukibean epicotyls resulted in alterations of the direction of newly deposited microfibrils, on the cell walls. Cells having transverse microfibrils on the inner surface of the wall were observed more frequently in GA₃-treated epicotyls than in untreated or water-treated ones. This effect of GA₃ was negated by simultaneously supplied colchicine. A crossed polylamellate structure was observed in the inner portion of the walls of GA₃-treated cells, but not in the inner portion of the walls of colchicine-treated cells. The wall formed under the influence of colchicine consisted of microfibrils running in the same direction.Abbreviations GA gibberellin - GA₃ gibberellic acid (gibberellin A₃)     

Probing single-cell micromechanics in vivo: the microrheology of C. elegans developing embryos

Cells are not directly accessible in vivo and therefore their mechanical properties cannot be measured by methods that require a direct contact between probe and cell. Here, we introduce a novel in vivo assay based on particle tracking microrheology whereby the extent and time-lag dependence of the mean squared displacements of thermally excited nanoparticles embedded within the cytoplasm of developing embryos reflect local viscoelastic properties. As a proof of principle, we probe local viscoelastic properties of the cytoplasm of developing Caenorhabditis elegans embryos. Our results indicate that unlike differentiated cells, the cytoplasm of these embryos does not exhibit measurable elasticity, but is highly viscous. Furthermore, the viscosity of the cytoplasm does not vary along the anterior-posterior axis of the embryo during the first cell division. These results support the hypothesis that the asymmetric positioning of the mitotic spindle stems from an asymmetric distribution of elementary force generators as opposed to asymmetric viscosity of the cytoplasm.     

Precision of nanoindentation protocols for measurement of viscoelasticity in cortical and trabecular bone

Nanoindentation has recently gained attention as a characterization technique for mechanical properties of biological tissues, such as bone, on the sub-micron level. However, optimal methods to characterize viscoelastic properties of bones are yet to be established. This study aimed to compare the time-dependent viscoelastic properties of bone tissue obtained with different nanoindentation methods. Bovine cortical and trabecular bone samples (n=8) from the distal femur and proximal tibia were dehydrated, embedded and polished. The material properties determined using nanoindentation were hardness and reduced modulus, as well as time-dependent parameters based on creep, loading-rate, dissipated energy and semi-dynamic testing under load control. Each loading protocol was repeated 160 times and the reproducibility was assessed based on the coefficient of variation (CV). Additionally, three well-characterized polymers were tested and CV values were calculated for reference.The employed methods were able to characterize time-dependent viscoelastic properties of bone. However, their reproducibility varied highly (CV 9–40%). The creep constant increased with increasing dwell time. The reproducibility was best with a 30 s creep period (CV 18%). The dissipated energy was stable after three repeated load cycles, and the reproducibility improved with each cycle (CV 23%). The viscoelastic properties determined with semi-dynamic test increased with increase in frequency. These measurements were most reproducible at high frequencies (CV 9–10%). Our results indicate that several methods are feasible for the determination of viscoelastic properties of bone material. The high frequency semi-dynamic test showed the highest precision within the tested nanoindentation protocols.     

A method for measuring linearly viscoelastic properties of human tympanic membrane using nanoindentation

A viscoelastic nanoindentation technique was developed to measure both in-plane and through-thickness viscoelastic properties of human tympanic membrane (TM). For measurement of in-plane Young's relaxation modulus, the TM sample was clamped on a circular hole and a nanoindenter tip was used to apply a concentrated force at the center of the TM sample. In this setup, the resistance to nanoindentation displacement can be considered due primarily to the in-plane stiffness. The load-displacement curve obtained was used along with finite element analysis to determine the in-plane viscoelastic properties of TM. For measurements of Young's relaxation modulus in the through-thickness (out-of-plane) direction, the TM sample was placed on a relatively rigid solid substrate and nanoindentation was made on the sample surface. In this latter setup, the resistance to nanoindentation displacement arises primarily due to out-of-plane stiffness. The load-displacement curve obtained in this manner was used to determine the out-of-plane relaxation modulus using the method appropriate for viscoelastic materials. From our sample tests, we obtained the steady-state values for in-plane moduli as approximately 17.4 MPa and approximately 19.0 MPa for posterior and anterior portions of TM samples, respectively, and the value for through-thickness modulus as approximately 6.0 MPa for both posterior and anterior TM samples. Using this technique, the local out-of-plane viscoelastic modulus can be determined for different locations over the entire TM, and the in-plane properties can be determined for different quadrants of the TM.