目的 制备聚乳酸(PLLA)三维微小凹图式并实现图式上新生大鼠海马神经干细胞(NSC)三维聚集体的形成及培养.方法 用2只新生1～3 dSPF级SD大鼠体外分离提取海马组织并培养NSC,观察其悬浮神经球和贴壁生长特性;以紫外光光刻和复制模塑法制备3种不同结构尺寸的PLLA微小凹图式;以普通光学显微镜和ImageJ图像处理软件研究图式上NSC聚集体的形成及大小分布;以免疫细胞荧光技术研究图式上NSC标志物Nestin的表达.结果 NSC在体外能够以悬浮神经球和贴壁生长的2种方式生长.在所研究的3种结构尺寸的图式中,无通道(120-0 μm)和通道宽度为20μm(120-20 μm)的微小凹图式更利于NSC聚集体的形成(P＜0.01);而在通道宽度为40 μm的图式(120-40 μm)上NSC更倾向于沿通道方向生长、迁移,并形成准一维排布的三维神经细胞网络.图式上聚集体Feret's直径分布均匀,无大的聚集体(直径＞ 120 μm)出现.在细胞聚集体及神经细胞网络形成期间NSC均保持Nestin阳性表达.结论 PLLA微小凹图式可用于体外NSC聚集体形成及神经细胞网络的三维图式化,这为干细胞组织工程及基于神经细胞的微系统提供了有效的研究及应用途径.

Tetrandrine is one of the major active ingredients in Menispermaceae Stephania tetrandra S. Moore, and has specific therapeutic effects in ischemic cerebrovascular disease. Its use in vascular dementia has not been studied fully. Here, we investigated whether tetrandrine would improve behavioral and cellular impairments in a two-vessel occlusion rat model of chronic vascular dementia. Eight weeks after model establishment, rats were injected intraperitoneally with 10 or 30 mg/kg tetrandrine every other day for 4 weeks. Behavioral assessment in the Morris water maze showed that model rats had longer escape latencies in training trials, and spent less time swimming in the target quadrant in probe trials, than sham-operated rats. However, rats that had received tetrandrine showed shorter escape latencies and longer target quadrant swimming time than untreated model rats. Hematoxylin-eosin and Nissl staining revealed less neuronal necrosis and pathological damage, and more living cells, in the hippocampus of rats treated with tetrandrine than in untreated model rats. Western blot assay showed that interleukin-1β expression, and phosphorylation of the N-methyl-D-aspartate 2B receptor at tyrosine 1472, were lower in model rats that received tetrandrine than in those that did not. The present findings suggest that tetrandrine may be neuroprotective in chronic vascular dementia by reducing interleukin-1β expression, N-methyl-D-aspartate receptor 2B phosphorylation at tyrosine 1472, and neuronal necrosis.

Direct and indirect evidence, in support of the notion that biological activity of three-dimensional (3-D) cultures may more closely mirror what happens in vivo, has appeared in the literature for the past three decades. This is probably best exemplified in the field of experimental oncology that adopts 3-D multicellular tumor spheroids (MCTS) to mimic the in vivo situation.

制备了微柱名义直径为4 μm或10 μm,名义间距为4μm或7μm,名义高度为4μm的聚二甲基硅氧烷微柱阵列型拓扑结构基底,研究了HepG2细胞与拓扑结构基底复合后细胞瞬时受体电位通道TRPV1、TRPV4在基因和蛋白水平的表达及其功能响应性.细胞TRPV1和TRPV4在基因水平表达的评价采用定量PCR技术进行;TRPV1和TRPV4在蛋白水平的表达以免疫印迹和免疫荧光染色确认;TRPV1和TRPV4功能响应性的研究系以TRPV1和TRPV4激动剂辣椒素和4α-佛波醇-12,13-二葵酸酯刺激细胞,采用钙离子染料钙绿-1结合激光共聚焦显微技术记录钙内流动态过程,以钙内流荧光响应幅度及阳性响应比率进行评价.实验结果表明,在四种拓扑结构基底上细胞TRPV1和TRPV4的mRNA表达量均显著高于平面基底上相应值.免疫印迹实验证实了TRPV1和TRPV4在蛋白水平的表达,且拓扑结构基底上TRPV1和TRPV4免疫荧光染色强度较之平面基底相应值明显增高或趋于增高.在激动剂作用下,TRPV1介导的钙内流表现为快速去敏感化(25秒内)的瞬态内流,且拓扑结构基底上阳性响应细胞比例或相对荧光响应幅度较之平面基底相应值增高;而拓扑结构基底上细胞TRPV4阳性响应细胞比例和相对荧光响应幅度较之平面基底均全面明显升高.上述结果表明,TRPV介导的离子信号可能是基底拓扑结构优化HepG2细胞功能表型的重要信号机制.

We have developed a polydimethylsiloxane (PDMS) pattern with arrays of microwells for the formation of multicellular aggregates by C17.2 neural stem cells. Upon interfacing with the patterns, the neural stem cells would firstly attach to the microwell sidewalls, forming cellular strips on day 1 after plating. For channel connected microwells, cellular strips on the concave semi-cylindrical sidewall surfaces continued among wells and through channels, followed by strip peeling due to prestress arising from actin filaments and assembly of suspending cellular aggregates within the microwells in the following 1-2 days. Our results also suggested that a small microwell diameter of 80 and 100 μm and a narrow channel width of 20 μm would facilitate the aggregate formation among the structural dimensions tested. Finite element method (FEM) simulation revealed that cellular strips on the semi-cylindrical sidewall surfaces peeled under significantly smaller prestresses (critical peeling prestress, CPP), than cells on flat substrates. However, the CPP by itself failed to fully account for the difference in aggregate inducing capability among the patterns addressed, suggesting cell growth behaviors might play a role. This study thus justified the current patterning method as a unique and practical approach for establishing 3D neural stem cell-based assay platforms.

在肿瘤及相伴血管生长过程中,微环境中的多种理化因素协同地发挥着重要的作用.传统体外实验多借助于Transwell等模型,在单一因素下考察细胞生物学效应,并不能反映在体的多因素微环境.基于微流控技术,本文构建了一种新的多细胞共培养模型,整合了多环境维度(二维/三维)、细胞与细胞及细胞与胞外基质相互作用、不同生化因子的浓度梯度、细胞区域性等多个重要因素,形成微环境,并能实时监测细胞的迁移和侵袭等响应.为评价该模型的可行性和功能上的独特优势,我们模拟了肿瘤细胞(HepG2,CAOV-3)和人脐静脉内皮细胞(HUVECs)共存的三维微环境,考察了它们共培养时相互诱导向三维基质材料中的迁移情况.结果表明,在三维共培养模型中细胞能够相互影响并出现明显形态差异;2种肿瘤细胞的诱导均使HUVECs迁移能力显著提高;同时2种不同肿瘤细胞出现了与其病理特质(HepG2低浸润,CAOV-3高浸润)相对应的迁移能力差异.以上结果表明,该模型可望为研究肿瘤微环境下的相关问题提供一个相对简便且更具整合价值的研究平台.

All living cells possess electrical characteristics and are thus responsive to, and even generate electric fields and currents. It has been shown that the electrical properties of cancer cells differ from normal proliferating cells, thus electric fields may induce differential effects in normal and cancer cells. Manipulation of these electrical properties may provide a powerful direct and/or adjuvant therapeutic option for cancer. A whole cell impedance-based biosensor to monitor the effects of a range of different frequencies (50 kHz–2 MHz) at low-intensity (<2 V/cm) on the growth rate of human SKOV3 ovarian cancer cells versus non-cancerous HUVECs is reported. Rapid real-time monitoring of the SKOV3 behavior was observed as the alternating electric fields were applied and the impedimetric response of the cells was recorded. The cells were also labeled with propidium iodide to examine morphological changes and cell viability with fluorescence microscopy with trypan blue for comparison. A noticeable decrease in the growth profile of the SKOV3 was observed with the application of 200 kHz alternating electric fields indicating specific inhibitory effects on dividing cells in culture in contrast to the HUVECs. The outcome of this research will improve our fundamental understanding of the behavior of cancer cells when exposed to alternating electric fields at specific frequencies and foster the development strategies and optimal parameters for alternating electric field therapies for clinical and drug delivery applications.

INTRODUCTION:In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs).METHODS AND MATERIALS:SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells' location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells' VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium Green™-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY.RESULTS:It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K(+). This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness.CONCLUSION:These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.

To establish vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) as secretary biomarkers for cell growth on topographic substrates, we have evaluated the secretion and expression of these 2 factors by SH-SY5Y human neuroblastoma cells on poly-L-lactide (PLLA) micropillar arrayed topographic substrates. We fabricated topographic substrates with UV lithography, silicon etching and polydimethylsiloxane-based replica molding, and interfaced SH-SY5Y human neuroblastoma cells with both the topographic substrates and PLLA flat substrates. Cell morphology and spreading were examined with scanning electron microscopy. The secretion and mRNA expression of VEGF and IL-8 were evaluated with enzyme linked immunosorbent assay (ELISA) and real time qPCR, respectively, 24 hours after cell plating. We successfully achieved 4 topographic substrates with a nominal pillar diameter of 2 microm and 4 microm, and a nominal pillar spacing of 2 microm and 7 microm. We found that the secretion and mRNA expression of VEGF and/or IL-8 by SH-SY5Y cells on 2-2 microm (pillar diameter-spacing), 4-2 microm and 4-7 microm topographic substrates were upregulated in comparison to those by cells on PLLA flat substrate, 24 hours after cell plating. Furthermore, both cytokines were even more substantially upregulated on the 2-7 microm substrate than on the other 3 topographic substrates. Compared to those on PLLA flat substrate, cells on topographic substrates showed significant changes in morphology (spreading area, perimeter and roundness), and the increase in the secretion and mRNA expression of VEGF and IL-8 was accompanied with a decrease in cell spreading areas. These results provided evidence that pillar arrayed topography was an important microenvironmental factor in affecting VEGF and IL-8 expression or secretion, and VEGF and IL-8 might serve as important secretary biomarkers for growth on topographic substrates by SH-SY5Y cells.

基于硅蚀刻及软光刻复制模塑技术,制备了4种聚二甲基硅氧烷(PDMS)微柱阵列型拓扑结构基底,其微柱名义直径为4μm或10μm,名义间距为4μm或7μm,名义高度为4μm.考察了人肝癌细胞株HepG2在拓扑结构基底与PDMS平面基底上的形态、生物合成和解毒相关分子标志物的分泌/表达.采用扫描电镜和相差显微图像分析发现,HepG2细胞接种于拓扑结构基底较之于平面基底上出现明显的形态铺展和极化程度变化.以ELISA检测显示,HepG2细胞在拓扑结构基底上的白蛋白分泌水平高于平面基底上的相应分泌量.用实时荧光定量PCR评价相关基因表达,发现HepG2细胞于拓扑结构基底上培养1～3天后,白蛋白、细胞色素P450第一家族A亚族蛋白多肽1和2(CYP1A1和CYP1A2)的mRNA表达量,高于平面基底上的相应表达值.在所研究结构尺寸范围的拓扑结构基底上,HepG2细胞较大的铺展面积和细胞圆度值以及较小的细胞长/短径比值伴随着较高的白蛋白分泌/表达量;较小的铺展面积和细胞圆度值以及较大的细胞长/短径比值却伴随着较高的CYP1A1和CYP1A2表达量.这些结果表明,基底拓扑结构是影响HepG2细胞形态及功能的重要微环境因素,并可成为基于肝细胞的反应器与微系统培养环境设计和细胞功能表型优化的有效工程化手段.

UV photolithography and hydrofluoric acid wet etching were used to produce silicon master molds and polydimethylsiloxane (PMDS)-based soft lithography was adopted to fabricate three-dimensional poly(lactic-co-glycolic acid) (PLGA) and PDMS microwell patterns with high aspect ratio and channel connection. Nine microwell patterns were thus obtained with different structural dimensions. Patterns were treated with oxygen plasma etching and polylysine coating to enhance hydrophilicity and cell compatibility for subsequent culture of C17. 2 neural stem cells. With proliferation during the culture, C17. 2 cells gradually distributed within the microwells, showing an obviously three-dimensional (3-D) growth behavior. The presence of channel structures greatly favored the 3-D growth of C17. 2 neural stem cells on the microwell patterns. Multi-layered scanning with confocal microscopy and 3-D rendering after carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining showed that most C17. 2 cells grew within a range of 30 to 90 microm from the microwell bottom. Immunofluorescence staining indicated that C17. 2 cells within 3-D microwell patterns were uniformly nestin-positive on day 2 after cell plating. It could well be concluded that the microwell patterns thus fabricated were suitable for the 3-D culture and subsequent differentiation of C17. 2 neural stem cells. And the cells can be maintained with uniform stemness properties while cultured in these microwell patterns.

UV lithography,silicon etching and soft lithography were adopted to fabricate three-dimensional poly-Llactide(PLLA) microwell patterns.Human neuroblastoma(SH-SY5Y) cells were integrated with the PLLA patterns,and then cell growth behaviors and distribution were evaluated.Voltage-gated calcium channels(VGCCs) response of SH-SY5Y cells cultured in microwells and on flat PLLA substrates was detected by confocal microscopy with a calcium fluorescent indicator(Calcium Green-1,AM).It was found that SH-SY5Y cells distributed on patterns's top surface showed two-dimensional(2-D) adhesion behavior,while those cells attached to the microwell's side-wall and bottom surface showed three-dimensional(3-D) and boundary two-dimensional(B2-D) adhesion behaviors,respectively.In response to 50 mmol/L high K + depolarization,the percentage of 2-D cells on PLLA flat substrates with positive VGCCs response was 91.2%,while this percentage was 75% and 81%,for 3-D and B2-D cells,respectively,significantly lower than that for 2-D cells.These results suggested that cell adhesion behaviors might be critical factors in affecting VGCCs responsiveness of SH-SY5Y cells.Microwell patterns could be an effective way for engineering 3-D microenvironment for neural cell culture and establishing 3-D cell-based biosensing platforms.

The present paper was aimed to explore the effect of Shuxuetong on the membrane viscoelasticity of erythrocyte taken from the acute phase patients suffering from chronic pulmonary heart disease. The membrane viscoelasticity of erythrocyte was taken from the acute phase patients suffering from chronic pulmonary heart disease. The changes of membrane viscoelasticity of erythrocyte after treated with shuxuetong were detected by micropipette aspiration technique. The results showed that the Shuxuetong of certain concentration could cause the decrease of membrane elastic modulus and viscous coefficients in acute phase patients suffering from chronic pulmonary heart disease. The study offers experimental evidences that the comprehensive treatment of pulmonary heart disease should involve the drug or measure to improve the erythrocyte deformability.

J. Neurochem. (2012) 122 , 374–381. Abstract Alzheimer’s disease (AD) affects about 35.6 million people worldwide, and if current trends continue with no medical advancement, one in 85 people will be affected by 2050. Thus, there is an urgent need to develop a cost‐effective, easy to use, sensor platform to diagnose and study AD. The measurement of peptide amyloid beta (Aβ) found in CSF has been assessed as an avenue to diagnose and study the disease. The quantification of the ratio of Aβ1–40/42 (or Aβ ratio) has been established as a reliable test to diagnose AD through human clinical trials. Therefore, we have developed a multiplexed, implantable immunosensor to detect amyloid beta (Aβ) isoforms using triple barrel carbon fiber microelectrodes as the sensor platform. Antibodies act as the biorecognition element of the sensor and selectively capture and bind Aβ1–40 and Aβ1–42 to the electrode surface. Electrochemistry was used to measure the intrinsic oxidation signal of Aβ at 0.65 V (vs. Ag/AgCl), originating from a single tyrosine residue found at position 10 in its amino acid sequence. Using the proposed immunosensor Aβ1–40 and Aβ1–42 could be specifically detected in CSF from mice within a detection range of 20–50 nM and 20–140 nM respectively. The immunosensor enables real‐time, highly sensitive detection of Aβ and opens up the possibilities for diagnostic ex vivo applications and research‐based in vivo studies.

UV lithography,silicon etching and soft lithography were adopted to fabricate micropillar arrayed cell culture substrates.With this approach,poly(dimethylsiloxane)(PDMS)topographic substrates with pillar featuring dimensions equal to or larger than 4 μm could be susccessfully fabricated,with a pillar height of 4 μm.The fabrication of substrates with a pillar feafuring dimension of 2 μm might represent the maximal fabrication capability of this method,as pillar lodging or missing could be found.With a simple tilting angle method,we fabricated a topographic substrate formed with a packed polystyrene microbead array.The arrayed beads could be as small as 2 μm in diameter or even in submicrometer range.Cell culture experiments showed that arrayed topographical substrates can promote adhesion or spreading,affecting resting membrane potential establishement and enhance the responsiveness of voltage-gated calcium channels of neuronal cells.The arrayed topographic substrates provide an efficient tool for engineering the microenvironments for sensor neuronal cells.

Objective: To compare the quantitative changes of the geometric shape of erythrocyte from the acute phase patients with chronic pulmonary heart disease between before and add Shuxuetong.Methods: In the venous blood 40 acute phase patients with chronic pulmonary heart disease between before and add drug,the ratio of erythrocyte surface/volume and sphericity index were determined using micropipette aspiration technique.Results: The acute phase patients with chronic pulmonary heart diseases were in contrast to normal human,the erythrocyte surface/volume ratio significantly lower,while sphericity index was significantly increased.In a certain concentration of Shuxuetong role,the red blood cell surface area / volume ratio(S / V) significantly increased,sphericity index was significantly lower.Conclusions: Shuxuetong of constant concentration can cause the decrease of the spherical indexes of erythrocytes in acute phase patients with chronic pulmonary heart disease.The study offers experimental evidences that the comprehensive treatment of pulmonary heart disease should involve the drug or measure to improve the erythrocyte deformability.

In this study, we applied specific blocking antibodies for integrin α6 or β1 subunit, and evaluated the in vitro effects of integrins α6β1 on the adhesion, chemotaxis and migration of hepatocellular carcinoma (HCC) cell line SMMC-7721 to type IV collagen. The adhesion force and cell migration, as measured by a micropipette aspiration system and Boyden chamber assay respectively, was dramatically reduced when either integrin subunits was blocked. The chemotaxis, as determined using a dual-micropipette system, was only affected by the antibody against β1 subunit. This study suggests that integrin α6β1 is an important cell surface receptor that mediates the adhesion of SMMC-7721 to type IV collagen. But the α6 subunit has minimal effect on pseudopod formation in response to type IV collagen. Therefore, the integrin α6β1-mediated cell migration is, at least in part, through the regulation on the cell adhesion step.

A micropipette technique was adopted to address the viscoelasticity and its relevance to microtubules and intermediate filaments in rat hepatocytes by treatment with colchicine (Col), acrylamide (Aery) or combination of both. The results showed that: under the concentrations studied, treatment with Aery, Col or combination of both agents resulted in a significant decrease in cell viscoelasticity. Analysis also indicated that microtubules might be more relevant to cell elasticity than intermediate filaments, as evidenced by changes in the principal elastic coefficient K1. The interaction between microtubules and intermediate filaments was discussed in terms of cell viscoelasticity for hepatocytes and hepatoma cells.

Objective: To explore the influence of integrin redistribution on hepatoma cell alignment and migration and the influence of cytoskeleton reassembly on integrin redistribution by the method of mechanical loading-unloading and fibronection(FN) coating. Method: By using immuneofluescence staining, confocal laser scanning microscopy and quantitative morphological analysis, integrin distribution change and crtoskeleton assembly adjustment were observed and the deformation of cell movement was tested and analyzed quantitatively. Results: (1) cells with different forms have different integrin expressions and distribution features. The β1 integrin expression for spreading cells was higher than that for round (nonspreading) cells. For spreading cells, the strongest staining was found towards the attachment surface. While for round (nonspreading) cells, the integrin staining on the free surface towards medium was stronger than that towards the attachment surface. (2) After 5 hours of mechanical stretch, the β1 integrin expression for both spreading and round cells increased, and distribution peaks towards the attachment surface broadened. At 1 hour after unloading, the β1 integrin expression decreased and the distribution of integrin staining showed the tendency of dispersion, especially for round cells. (3) After coating the substrates with FN, the β1 integrin expression increased. The integrin staining for either spreading or round cells was more towards the attachment surface to reduce the migration of hepatoma cells. (4) After 5 hours of mechanical stretch, 60% of cells showed their orientation of major axes distributed between 70°~110° towards the stretching direction, and the cytoskeleton aligned vertically to the stretching direction. Cytoskeletons were found significantly depolymerized at 1 hour of unloading. Conclusions: The change of integrin distribution is affected by cytoskeleton aligned and the number of ligand. The distribution feature of the whole integrin expression on the surface of individual round cells is related to their stronger invasion and metastasis capability.

Objective To compare the growth behaviors of rat hippocampal neural stem cells (NSCs) cultured in 2 medium conditions to optimize the proliferation condition, and to achieve integration of hippocampal NSCs with our fabricated three-dimensional (3-D) poly-L-lactide (PLLA) microwell patterns. Methods Rat hippocampal cells, obtained with mechanical separation, were expanded in either Neurobasal- or DMEM/F12-based culture medium. Proliferation behaviors of the isolated hippocampal cells were evaluated with neural sphere counting and the methylthiazolyl tetrazolium (MTT) assay. PLLA microwell patterns were fabricated with UV lithography, silicon etching and soft lithography and then integrated with the expanded NSCs. Results Immunocytochemistry and immunofluorescence showed that the isolated hippocampal cells were positive for neural stem cell marker nestin and were capable of differentiating to the neuronal and glial lineages. In a period of 30 days’ expanding, hippocampal NSCs proliferated slowly forming neural spheres in Neurobasal-based culture medium, with few cells attached to substrates and differentiated morphologically. In contrast, cells in DMEM/F12-based medium proliferated rapidly, with significant cell attachment and morphological differentiation. On the day 25 of expanding, neural sphere number in DMEM/F12-based medium was 4.7 times as much as that in Neurobasal-based medium. The microwell patterns were well defined, of high aspect ratio (≥1) and stable under culture conditions. Population expanded hippocampal NSCs were successfully integrated with the 3-D PLLA microwell patterns. Conclusion DMEM/F12-based medium is favorable to population expansion of rat hippocampal cells while Neurobasal-based medium is conducive to purification of hippocampal NSCs. Sequential use of DMEM/F12- and Neurobasal-based medium can thus result in expansion of hippocampal NSCs and successful integration of the NSCs with 3-D PLLA microwell patterns.