结合AFM探测副伤寒沙门氏菌B感染红细胞膜的生物力学特性

采用原子力显微镜和衍射显微术,在纳米精确尺度探测副伤寒沙门氏菌B(Sp B)感染宿主红细胞(RBC)膜微观结构和力学特性,涉及细胞的形变、膜面内剪切模量和弯曲模量.结合这两种单分子测量技术,利用相关的数学模型表述RBC膜对菌体Sp B的入侵非常敏感.实验结果显示,不同感染期间的Sp B寄生菌体,能够引起宿主RBC膜结构改变,形变能力降低,膜剪切模量和弯曲模量显著增加.这些力学特性的变化影响RBC的输氧和循环功能.实验结果表明,Sp B具有独特的鞭毛调控系统,入侵的毒性菌体寄生蛋白与血影蛋白网络中的运输蛋白有特异结合位点,导致RBC膜骨架网络、波动力学和细胞内、外基质都产生应激反应,这有可能为理解Sp B感染RBC的发病机理和寄生途径提供一些新的实验思路和分析依据.     

基于图像分析技术的红细胞膜力学特性多参数动态测定的研究

探索以图像分析技术,在无扰、在位、实时的情况下,对单个活态红细胞的多个力学参量：弯曲模量KC、剪切模量μ及切向与弯曲模量之比ε等进行非侵入性连续动态测定的新方法。以该技术对红细胞在不同外部条件（温度、氧分压、渗透压）下的力学参量进行动态监测,不但揭示出有关变量条件对细胞各个力学参量的影响。还证明了本技术适于对细胞的各种生理和病理过程进行连续监测。     

柯萨奇病毒B组5型非结构蛋白抑制NF-κB信号通路的作用机制研究

目的 柯萨奇病毒B组5型（CVB5）是手足口病的重要病原体之一,可导致发热、皮疹或疱疹等临床症状,重症者出现神经系统疾病,甚至死亡。天然免疫应答是机体抗病毒入侵的第一道防线,其中核因子κB (NF-κB)是宿主天然免疫反应中的重要蛋白质,然而关于CVB5感染后调控NF-κB介导信号通路的研究尚鲜有报道。方法 本研究通过检测启动子活性、促炎因子水平以及通路中关键蛋白表达等,阐明CVB5对NF-κB信号通路的调控作用机制。结果 CVB5感染可抑制促炎因子表达和p65的磷酸化。CVB5非结构蛋白（NSP）可抑制促炎因子表达以及重要蛋白p65和IκBα的磷酸化。经STRING11.1数据库预测表明,CVB5 3CD蛋白与宿主多聚胞嘧啶结合蛋白1 (PCBP1)具有相互作用,且PCBP1可促进IκBα和p65的磷酸化,抑制病毒复制。结论 CVB5 NSP可负调控NF-κB信号通路,且与3CD相互作用的PCBP1蛋白可通过调控NF-κB通路抑制CVB5复制。本研究探索病毒与宿主天然免疫应答的调控作用,从而为研制抗CVB5感染的药物提供作用靶点。     

寄生对集团内捕食系统中物种入侵的影响

寄生已被证明是影响生物入侵动态的关键因素,可通过直接或间接作用影响物种间相互作用。基于元胞自动机模型,探讨了寄生感染对宿主种群的间接作用(寄生对宿主的密度调节和特征调节效应)在外来种入侵动态中的作用。结果显示,寄生对宿主的3种调节效应均对外来种的入侵起到了抑制作用;同时,相对于密度调节效应,寄生的特征调节对捕食者种群入侵的抑制作用更明显,而两者的耦合调节效应对入侵的抑制能力明显高于单种效应的作用。综上,该研究在一定意义上丰富和发展了生物入侵的理论,为生物入侵的有效控制提供了理论参考。     

肺炎链球菌粘附机制的研究现状

肺炎链球菌粘附宿主我肺炎链球菌侵袭、感染宿主细胞的先决条件。粘附过程是特异的,是细菌表面的粘附分子和宿主细胞膜受体相互作用的结果。英膜对肺炎链球菌的粘附无影响,而细胞壁（ＣＷ）在介导肺炎链附粘附宿主细胞过程中起重要作用;ＣＷ亚组人脂磷酸（ＬＴＡ）介导肺炎链球菌的粘附过程,并导致炎症反应;细菌表面的结构蛋白或分泌蛋白是细菌与宿主细胞连接的桥梁;肺炎链球菌能与宿主细胞外基质蛋白特异性结合,进而粘附宿主     

拟菌体包囊膜的动态变化和宿主细胞与细菌的相互关系

本文初次报道紫云英根瘤的超微结构。用根瘤中段的中心组织作实验材料,以显示受根瘤菌侵染的宿主细胞的一般结构。细菌借助于侵入线进入宿主细胞,发育成拟菌体,为包囊膜所裹。一个包囊膜内一般只有一个拟菌体。包囊膜可以与细胞质内的囊泡和小液泡融合而扩增,导致膜对拟菌体的包裹由紧密到疏松的变化。包囊膜和拟菌体表面都有突起,两者的突起相对接触和融合。对拟菌体包囊膜的动态变化与衰老的关系以及宿主细胞和拟菌体之间物质交换的关系进行了讨论。作者指出包囊膜的扩增和电子透明区域的存在,是拟菌体发育成熟的一个阶段,包囊膜和拟菌体通过互相突起、融合沟通的结构,可能是宿主细胞和细菌之间物质交换功能的一种表现。     

InlA和InlB介导单核细胞增生李斯特菌入侵宿主细胞分子机制的研究进展

单核细胞增生李斯特菌(Listeria monocytogenes)是一种革兰氏阳性食源性致病菌。在造成宿主食源性感染的过程中, 单核细胞增生李斯特菌能凭借其独特的表面蛋白入侵宿主的非吞噬细胞。内化素蛋白家族(Internalins)是介导单核细胞增生李斯特菌入侵宿主非吞噬细胞的主要因子。本文根据国内外一些最新的研究成果, 结合作者近几年的工作, 综述了在侵染宿主的过程中, 单核细胞增生李斯特菌主要的内化素蛋白InlA和InlB介导细菌入侵宿主细胞的分子机制, 以期为阐明食源性致病菌致病机理、预防和治疗食源性疾病提供理论基础。     

鲻鱼寄生蠕虫种群和群落组成分析

从1018尾鲻鱼（Mugil cephalus L．）体内获得各类寄生蠕虫9种,共2133个虫体,其中复殖吸虫8种,棘头虫1种。寄生蠕虫总的感染率为24．4％,感染强度1－81,平均感染强度为8．8,平均密度为2．3。鲻长尾吸虫（Elonginus mugilus）和双睾原单脏吸虫（Prohaplanchnus diorchis）的感染率随着宿主体长增加而下降,而楔形单脏吸虫（Haplosplanchnus cuneatus）、长形单脏吸虫（H．elongatus）、浦里单脏吸虫（H．purii）、惠东拟囊腔吸虫（Saccocoelioides huidongensis）、粤东纵睾吸虫（Tandemorchis yuedongensis）、单睾椭宝贵虫（Elliptobursa singlorchis）和圆颚针鱼新棘吻虫（Neoechinorhynchus tylosuri）的感染率与宿主体长相关不显著（P＞0．05）。研究结果还表明,楔形单脏吸虫、长表单脏吸虫、惠东拟囊腔吸虫和浦里单脏吸虫的感染强度则随着宿主体长增加而增大,其余5种寄生蠕虫的感染强度与宿主体长相关性不显著（P＞0．05）,鲻鱼寄生蠕虫在不同月的感染率和种群数量差异,除10月与11月及8月与9月不明显外（P＞0．05）,其余月份之间的差异显著（P＜0．01）。根据感染率和种群密度变化分析,优势种为惠东拟囊腔虫;通过模糊聚类分析,将鲻鱼体内寄生蠕虫组分群落分为3个亚群,宿主体长小于70mm的寄生蠕虫亚组分群落的平均种数、优势度、感染指数、多蠕虫组分群落分为3个亚群,宿主体长小于70mm的寄生蠕虫亚组分群落的平均种数、优势度、感染指数、多样性指标、平均密度和感染强度相对较低;体长于70mm的亚组分群落下值则随体长增加而上升,而平均种数减少。     

病毒与细胞的微生态学研究进展

微生态学（microecology）是细胞水平或分子水平的生态学,它是研究微生物群的结构和功能,以及微生物与其宿主相互依赖、相互制约关系的科学。病毒（virus）是一种比细菌还要微小和简单的非细胞形态微生物（acellular microorganism）,不仅可寄生于动植物体的组织细胞表面或内部,还可寄生于细菌、真菌等微生物内部。作为宿主以及细胞内的寄生体,病毒除引起宿主多种类型的感染外,还可参与宿主组织细胞的微生态系的组成,赋予细胞干扰相关病毒增殖与复制、抵抗特定病毒感染感染的作用,并能引起宿主细胞产生特定毒素、获得新抗原性等改变。本研究通过微生态学角度,对病毒与细胞的相互关系及病毒与细胞微生态学在医学上的作用作一综述。     

冠状病毒S蛋白及其受体的结构和功能

冠状病毒是有包膜的单股正链RNA病毒。作为人和动物的重要致病原,冠状病毒感染主要导致宿主呼吸系统、肝脏、胃肠道以及神经系统出现急性或慢性症状。2000年以来,传染性非典型肺炎和中东呼吸综合征的暴发,以及猪流行性腹泻病毒在全球猪群中的暴发流行,引起大家对动物冠状病毒的极大重视。S蛋白具有受体结合活性和膜融合活性,是冠状病毒感染细胞的关键蛋白;S蛋白在病毒的组织或宿主嗜性和毒力等方面发挥重要作用。本文重点对近年来冠状病毒S蛋白的结构、功能以及S蛋白与受体相互作用的研究进行综述,以期为冠状病毒的入侵机制和反向遗传学研究以及受体阻断药物的开发提供参考。     

原子力显微镜技术检测几种骨组织细胞力学性能

为了探究几种骨组织细胞系的力学性能及其与细胞功能的关系,该文采用原子力显微镜压陷法分别检测了前成骨细胞系（2T3和MC3T3-E1）、前骨细胞系（MLO-A5）和骨样细胞系（MLO-Y4）的杨氏模量,利用激光共聚焦显微镜观察了这几种细胞微丝和微管的排布。结果显示,2T3、MC3T3-E1、MLO-A5和MLO-Y4细胞的杨氏模量分别为（7000±2015）Pa、（6600±2024）Pa、（4700±644）Pa和（4500±1622）Pa,与原代骨组织细胞的杨氏模量及变化趋势保持一致,但两种前成骨细胞的杨氏模量要显著高于前骨细胞和骨细胞。细胞荧光染色结果表日月＇前成骨细胞细胞核周围的微丝和微管分布密度要高于前骨细胞和骨细胞,而前骨细胞MLO-A5,尤其是骨细胞MLO-Y4的骨架主要集中于细胞突触和边缘,这可能是导致几种细胞力学性能差异的原因。该研究从生物力学的角度为进一步深入理解骨组织细胞结构与功能的关系提供了实验依据。     

Direct measurement of shear strain in adherent vascular endothelial cells exposed to fluid shear stress

Functional and morphological responses of endothelial cells (ECs) to fluid shear stress are thought to be mediated by several mechanosensitive molecules. However, how the force due to fluid shear stress applied to the apical surface of ECs is transmitted to the mechanosensors is poorly understood. In the present paper, we performed an analysis of an intracellular mechanical field by observation of the deformation behaviors of living ECs exposed to shear stress with a novel experimental method. Lateral images of human umbilical vein ECs before and after the onset of flow were obtained by confocal microscopy, and image correlation and finite element analysis were performed for quantitative analyses of subcellular strain due to shear stress. The shear strain of the cells changed from 1.06 ± 1.09% (mean ± SD) to 4.67 ± 1.79% as the magnitude of the shear stress increased from 2 to 10 Pa. The nuclei of ECs also exhibited shear deformation, which was similar to that observed in cytoplasm, suggesting that nuclei transmit forces from apical to intracellular components, as well as cytoskeletons. The obtained strain-stress relation resulted in a mean shear modulus of 213 Pa for adherent ECs. These results provide a mechanical perspective on the investigation of flow-sensing mechanisms of ECs.     

Theoretical model and experimental study of red blood cell (RBC) deformation in microchannels

The motion and deformation of red blood cells (RBCs) flowing in a microchannel were studied using a theoretical model and a novel automated rheoscope. The theoretical model was developed to predict the cells deformation under shear as a function of the cells geometry and mechanical properties. Fluid dynamics and membrane mechanics are incorporated, calculating the traction and deformation in an iterative manner. The model was utilized to evaluate the effect of different biophysical parameters, such as: inner cell viscosity, membrane shear modulus and surface to volume ratio on deformation measurements. The experimental system enables the measurement of individual RBCs velocity and their deformation at defined planes within the microchannel. Good agreement was observed between the simulation results, the rheoscope measurements and published ektacytometry results. The theoretical model results imply that such deformability measuring techniques are weakly influenced by changes in the inner viscosity of the cell or the ambient fluid viscosity. However, these measurements are highly sensitive to RBC shear modulus. The shear modulus, estimated by the model and the rheoscope measurements, falls between the values obtained by micropipette aspiration and laser trapping. The study demonstrates the integration of a theoretical model with a microfabricated device in order to achieve a better understanding of RBC mechanics and their measurement using microfluidic shear assays. The system and the model have the potential of serving as quantitative clinical tools for diagnosing deformability disorders in RBCs.     

透射电镜观察p21-（HBsAg/HBsAg）转基因小鼠肝癌细胞凋亡

目的观察p21HBsAg/HBsAg转基因小鼠肝癌细胞凋亡。方法用透射电镜观察p21HBsAg/HBsAg转基因小鼠肝癌细胞凋亡的形态学改变。结果细胞凋亡早期,细胞核染色体发生边集,核形不规整,核膜表面凹凸;凋亡中期,核内染色质凝聚,趋边呈月牙状,核膜孔消失,核膜呈波纹状皱缩;凋亡晚期,核固缩,细胞膜出芽形成小泡,可见凋亡小体。结论p21HBsAg/HBsAg转基因小鼠肝癌细胞凋亡具有典型性病变特征,是研究HBV感染诱发肝癌发病机理的合适动物模型。     

国产对囊蕨亚科（蹄盖蕨科）植物的管状分子

利用扫描电镜观察了国产蹄盖蕨科（Athyriaceae）对囊蕨亚科（Deparioideae）10种植物及双盖蕨属（Diplazium Sw．）3种植物根状茎的管状分子。结果显示,这些管状分子端壁和侧壁的形态及结构分别相同且侧壁具有穿孔板（多穿孔板）。根据穿孔板的形态特征,将该亚科的管状分子分为5种类型：（1）梯状穿孔板,无穿孔的二型性现象：（2）梯状穿孔板,有穿孔的二型性现象：（3）网状穿孔板：（4）梯状-网状混合的穿孔板：（5）大孔状穿孔板。按照纹孔膜残留的程度又可分为3种：部分区域有完整的纹孔膜、残留呈网状或线状以及很少或无纹孔膜残留。结合前人的研究资料,发现蕨类植物的管状分子与被子植物的导管分子在形态和输导机理上存在明显差异,管胞和导管分子不能仅仅根据纹孔膜的存在与否来确定,而应根据穿孔板存在于端壁还是侧壁进行判断,即穿孔板仅存在于端壁的管状分子为导管分子：端壁和侧壁形态及结构分别相同,有或无穿孔板的管状分子为管胞。由此可以推测蕨类植物和裸子植物中输导水分和矿物质的管状分子主要为管胞。单叶双盖蕨属（Triblemma（J．Sm．）Ching）与双盖蕨属管状分子的特征并不相似,显示了将单叶双盖蕨属从双盖蕨属独立出来归人对囊蕨亚科的合理性。根据管状分子的特征,推测假蹄盖蕨属（Athyriopsis Ching）和蛾眉蕨属（Lunathyrium Koidz．）可能是比较进化的属,而介蕨属（Dryoathyrium Ching）相对比较原始,单叶双盖蕨属的系统位置应介于假蹄盖蕨属与介蕨属之间。     

Computational Analysis of Dynamic Interaction of Two Red Blood Cells in a Capillary

The dynamic interaction of two red blood cells (RBCs) in a capillary is investigated computationally by the two-fluid model, including their deformable motion and interaction. For characterization of the deformation, the RBC membrane is treated as a curved two-dimensional shell with finite thickness by the shell model, and allowed to undergo the stretching strain and bending deformation. Moreover, a Morse potential is adopted to model the intercellular interaction for the aggregation behavior, which is characterized as the weak attraction at far distance and strong repulsion at near distance. For validation of the present technique, the dynamic interaction of two RBCs in static blood plasma is simulated firstly, where the RBCs aggregate slowly until a balanced configuration is achieved between the deformation and aggregation forces. The balanced configuration is in good agreement with the results reported previously. Three important effects on the dynamic behavior of RBCs are then analyzed, and they are the initial RBC shape, RBC deformability, and the intercellular interaction strength. It is found that the RBC is less deformed into a well-known parachute shape when the initial RBC shape is larger. Similarly, if the elastic shear modulus and bending stiffness of RBC membrane increase, the RBC resistance to deformation becomes higher, such that the RBC is less deformed. The simulation results also demonstrate that the RBC deformability strongly depends on the intercellular interaction strength. The RBCs deform more easily as the intercellular interaction strength increases.     

Exploring the mechanical properties of single vimentin intermediate filaments by atomic force microscopy

Intermediate filaments (IFs), together with actin filaments and microtubules, compose the cytoskeleton. Among other functions, IFs impart mechanical stability to cells when exposed to mechanical stress and act as a support when the other cytoskeletal filaments cannot keep the structural integrity of the cells. Here we present a study on the bending properties of single vimentin IFs in which we used an atomic force microscopy (AFM) tip to elastically deform single filaments hanging over a porous membrane. We obtained a value for the bending modulus of non-stabilized IFs between 300 MPa and 400 MPa. Our results together with previous ones suggest that IFs present axial sliding between their constitutive building blocks and therefore have a bending modulus that depends on the filament length. Measurements of glutaraldehyde-stabilized filaments were also performed to reduce the axial sliding between subunits and therefore provide a lower limit estimate of the Young's modulus of the filaments. The results show an increment of two to three times in the bending modulus for the stabilized IFs with respect to the non-stabilized ones, suggesting that the Young's modulus of vimentin IFs should be around 900 MPa or higher.     

Viscoelasticity of the human red blood cell

We report here the first measurements of the complex modulus of the isolated red blood cell (RBC). Because the RBC is often larger than capillary diameter, important determinants of microcirculatory function are RBC deformability and its changes with pathologies, such as sickle cell disease and malaria. A functionalized ferrimagnetic microbead was attached to the membrane of healthy RBC and then subjected to an oscillatory magnetic field. The resulting torque caused cell deformation. From the oscillatory forcing and resulting bead motions, which were tracked optically, we computed elastic and frictional moduli, g' and g', respectively, from 0.1 to 100 Hz. The g' was nearly frequency independent and dominated the response at all but the highest frequencies measured. Over three frequency decades, g' increased as a power law with an exponent of 0.64, a result not predicted by any simple model. These data suggest that RBC relaxation times that have been reported previously, and any models that rest upon them, are artifactual; the artifact, we suggest, arises from forcing to an exponential fit data of limited temporal duration. A linear range of response was observed, but, as forcing amplitude increased, nonlinearities became clearly apparent. A finite element model suggests that membrane bending was localized to the vicinity of the bead and dominated membrane shear. While the mechanisms accounting for these RBC dynamics remain unclear, methods described here establish new avenues for the exploration of connections among the mechanical, chemical, and biological characteristics of the RBC in health and disease. storage modulus; loss modulus; magnetic twisting cytometry; erythrocyte; viscoelasticity; rheology     

Membrane bending modulus and adhesion energy of wild-type and mutant cells of Dictyostelium lacking talin or cortexillins.

We have employed an interferometric technique for the local measurement of bending modulus, membrane tension, and adhesion energy of motile cells adhering to a substrate. Wild-type and mutant cells of Dictyostelium discoideum were incubated in a flow chamber. The flow-induced deformation of a cell near its adhesion area was determined by quantitative reflection interference contrast microscopy (RICM) and analyzed in terms of the elastic boundary conditions: equilibrium of tensions and bending moments at the contact line. This technique was employed to quantify changes caused by the lack of talin, a protein that couples the actin network to the plasma membrane, or by the lack of cortexillin I or II, two isoforms of the actin-bundling protein cortexillin. Cells lacking either cortexillin I or II exhibited reduced bending moduli of 95 and 160 k(B)T, respectively, as compared to 390 k(B)T, obtained for wild-type cells. No significant difference was found for the adhesion energies of wild-type and cortexillin mutant cells. In cells lacking talin, not only a strongly reduced bending modulus of 70 k(B)T, but also a low adhesion energy one-fourth of that in wild-type cells was measured.     

Alteration of red cell membrane viscoelasticity by heat treatment: effect on cell deformability and suspension viscosity

The membrane shear elastic modulus (mu) and the time constant for extensional shape recovery (tc) were measured for normal, control human red blood cells (RBC) and for RBC heat treated (HT) at 48 degrees C. Three separate methods for the measurement of mu were compared (two used a micropipette and one employed a flow channel), and the membrane viscosity (n) was calculated from the relation n = mu. tc. The deformability of HT and control cells was evaluated using micropipette techniques, and the bulk viscosity of RBC suspensions at 40% hematocrit was measured. The shear elastic modulus, or "membrane rigidity", was more than doubled by heat treatment, although both the absolute value for mu and the estimate of the increase induced by heat treatment varied depending on the method of measurement. Heat treatment caused smaller increases in membrane viscosity and in membrane bending resistance, and only minimal changes in cell geometry. The deformability of HT cells was reduced: 1) the pressure required for cell entry (Pe) into 3 micrometers pipettes was increased, on average, by 170%; 2) at an aspiration pressure (Pa) exceeding Pe, longer times were required for cell entry into the same pipettes. However, when Pa was scaled relative to the mean entry pressure for a given sample (i.e, Pa/Pe), entry times were similar for control and HT cells. Bulk viscosity of HT RBC suspensions was elevated by approximately 12% on average (shear rates 75 to 1500 inverse seconds). These findings suggest that alteration of RBC membrane mechanical properties, similar to those induced by heat treatment, would most affect the in vivo circulation in regions where vessel dimensions are smaller than cellular diameters.