细胞外基质对血管平滑肌细胞桩蛋白和纽蛋白表达的影响

【目的】探讨细胞外基质（extracellular matrix,ECM）对体外培养的血管平滑肌细胞（smooth muscle cells,SMCs）黏着斑（focal adhesions,FAs）分子桩蛋白（paxillin）和纽蛋白（vinculin）表达的影响。【方法】将第4～6代大鼠主动脉SMCs分别种植在涂有纤维连接蛋白（fibronectin,FN）或层黏连蛋白（laminin,LN）的盖玻片上或种植在基质胶内进行培养。采用共聚焦免疫荧光技术检测paxillin和vinculin的表达,并通过FAs分子paxillin／F-aetin和vineulin／F-actin的双重荧光染色显示。【结果】种植在FN上的vinculin表达最低,LN次之,而在Matrigel胶中表达最高;在FN上vinculin在胞质分布较多,在FA＇s较少;而生长在LN基质上和基质胶中vincu1in较多定位在FAs,在胞质分布较少。Vinculin的荧光强度在FN、LN及基质胶中分别为（64．72±7．18）AU／μm2,（89．82±5．11）AU／μm2,（117．19土16．99）AU／μm2,依次增高,且组间比较有统计学意义（P〈0．05）。种植在FN上的paxillin最高,LN上次之,而Matrigel胶中表达最低;基质胶中paxillin主要分布于FAs,而FN上的除分布于FAs外,在胞浆的表达水平也较高。Paxillin的荧光强度在FN、LN及基质胶中分别为（129．87±10．22）AU／μm2、（86．59±9．90）AU／μm2、（56．32±7.54）AU／μm2,依次降低,且组间比较有统计学意义（P〈0．05）。【结论】不同的ECM成分对FAs分子的表达有影响：FN上调paxillin的表达,下调vinculin的表达;而LN和基质胶上调vinculin的表达,下调paxillin的表达。     

选择性环氧合酶-2抑制剂通过Rho/ROCK通路对2型糖尿病合并非酒精性脂肪性肝炎大鼠肝脏保护作用机制的研究

目的:探讨选择性环氧合酶-2抑制剂塞来昔布是否通过抑制Rho/Rho相关蛋白激酶（ROCK）通路的表达对2型糖尿病（T2DM）合并非酒精性脂肪性肝炎（NASH）大鼠肝脏具有保护作用。方法:雄性SD大鼠40只,随机等分为T2DM合并非酒精性脂肪性肝炎（T2DM-NASH）组、T2DM-NASH+塞来昔布组、对照组、对照+...     

系统性红斑狼疮患者骨髓间质干细胞超微结构及细胞骨架的特征

目的 探讨系统性红斑狼疮(SLE)患者骨髓间质干细胞(MSCs)的超微结构及细胞骨架的形态学特征.方法 体外培养SLE患者的MSCs后,透射电子显微镜(TEM)、激光共聚焦显微镜(LCM)观察超微结构及肌动蛋白、纽蛋白的表达.结果 SLE MSCs透射电子显微镜下多数细胞内呈衰老征象,可见大量自噬体;细胞边缘应力纤维增多,排列紊乱,组成粗大的强荧光团,核周围应力纤维减少;纽蛋白在细胞边缘排列紊乱,细胞核周围增多.结论 SLE MSCs超微结构及细胞骨架的改变是细胞扩增不良的原因之一.     

三磷酸腺苷对人胃癌细胞恶性表型的去恶化作用

目的 观察三磷酸腺苷（ＡＴＰ）对人胃癌Ｍ１７细胞系增殖和分化的影响。方法 应用三磷酸腺苷作用于人胃癌Ｍ１７单细胞克隆亚系,每日记数细胞和计算抑制率,用扫描电镜观察Ｍ１７细胞膜表面微绒毛变化,罗氏黄荧光染料传输法观察细胞间隙连接通讯功能的状况,纽蛋白和微丝蛋白双标记法观察微丝骨架和粘着斑组装的变化。结果 （１）ＡＴＰ对人胃癌细胞的增殖有明显的抑制作用,加药处理４天后增殖的抑制率可达８０％以上。（２）     

Extracellular matrix regulates microfilament and vinculin organization in C6-glioma cells

Since the extracellular matrix (ECM) has a role in regulating cell proliferation it has been hypothesized that unregulated growth of transformed cells results from an inability of cells to interpret the constraints on growth as dictated by the ECM. The most likely candidate to communicate the restraint on growth to the nucleus is the cytoskeleton because it is the only cytoplasmic structure to physically connect the nucleus and plasma membrane. The purpose of this study was to determine whether the cytoskeleton, specifically the microfilaments (MF), of the C6-glioma cell line prossesses the ability to respond to changes in the ECM. The C6-glioma cell line was chosen as a model because it exhibits the characteristics of a tumor cell, both in vivo and in vitro. In this study cells were grown on bare glass, Matrigel, laminin and type IV collagen prior to staining with phalloidin tetramethylrhodamine B isothiocyanate or antibodies specific to vinculin to visualize MF and adhesion plaques, respectively. On the basis of MF orientation, network complexity and location of adhesion plaques, this study reports that the cytoskeletal arrangements of C6 cells grown on various substrates are distinctly different. Each distinct organization pattern may reflect a change of cell behavior promoted by the different culture conditions. The significance of this study is that it demonstrates that the process of transformation in C6-glioma cells has not interfered with the cells ability to recognize, interpret and adapt to changes in the ECM. Received: 5 July 1994 / Revised: 21 June 1995 / Accepted: 7 August 1995     

Time-dependent Changes in Smooth Muscle Cell Stiffness and Focal Adhesion Area in Response to Cyclic Equibiaxial Stretch

Observations from diverse studies on cell biomechanics and mechanobiology reveal that altered mechanical stimuli can induce significant changes in cytoskeletal organization, focal adhesion complexes, and overall mechanical properties. To investigate effects of short-term equibiaxial stretching on the transverse stiffness of and remodeling of focal adhesions in vascular smooth muscle cells, we developed a cell-stretching device that can be combined with both atomic force and confocal microscopy. Results demonstrate that cyclic 10%, but not 5%, equibiaxial stretching at 0.25 Hz significantly and rapidly alters both cell stiffness and focal adhesion associated paxillin and vinculin. Moreover, measured changes in stiffness and focal adhesion area from baseline values tend to correlate well over the durations of stretching studied. It is suggested that remodeling of focal adhesions plays a critical role in regulating cell stiffness by recruiting and anchoring actin filaments, and that cells rapidly remodel in an attempt to maintain a homeostatic biomechanical state when perturbed above a threshold value.     

钮蛋白在神经生长锥的三维分布及其机能意义

目的　探索钮蛋白 (vinculin)在神经生长锥形态变化和神经生长过程中的作用机制。　方法　用免疫细胞化学方法 ,检测了初代培养鸡胚脊神经节细胞生长锥的细胞骨架蛋白vinculin的三维分布特征及其与纤维型肌动蛋白 (F actin)的相互关系。　结果　在三维的水平面上 ,vinculin与F actin的免疫反应活性在P领域和C领域的部分区域重合 ,在P领域伸展方向前端和丝状伪足内免疫反应活性最强。vinculin比F actin更接近细胞膜分布 ;在纵轴方向 ,vinculin主要分布在中部以上的表面游离侧 ,基质侧未见分布 ;F actin大部分与vinculin重叠 ,但比vinculin分布更广泛。　结论　vinculin是构成膜骨架蛋白成分之一 ,具有连接生长锥细胞膜和F actin的作用 ,参与生长锥形态变化、伪足伸缩 ,使神经突起生长。     

同位素标记相对与绝对定量技术筛选出Vinculin蛋白在进展期胃癌组织中低表达及其预后意义

 目的 应用同位素标记相对与绝对定量蛋白组学（isobaric tags for relative and absolute quantification,iTRAQ）技术鉴定和筛选进展期胃癌复发转移相关的差异蛋白;检测筛选出的目标蛋白Vinculin表达水平及其预后意义。方法 应用iTRAQ技术在正常胃黏膜组织组、胃癌复发组和胃癌未复发组,检测差异表达的蛋白。然后选取差异性表达显著的Vinculin蛋白,采用免疫组织化学（immunohistochemistry,IHC）技术在110例人胃癌组织标本中检测其表达情况,并统计分析Vinculin蛋白表达量与临床病理学指标的关系;采用Kaplan-Meier生存曲线分析,探究Vinculin蛋白表达与胃癌患者生存期的关系。结果 Vinculin蛋白主要表达于细胞质中近胞膜处;在110例人胃癌组织标本中,Vinculin蛋白阳性表达46例（41.8%）,缺失表达64例（58.2%）。统计结果显示,Vinculin蛋白缺失表达与淋巴结转移（P=0.018）、肿瘤TNM分期（P=0.022）、血管侵袭（P=0.002）显著正相关。Kaplan-Meier生存曲线分析结果显示,Vinculin缺失表达与胃癌患者不良预后正相关（P=0.011）。结论 Vinculin蛋白缺失表达与肿瘤转移和预后不良相关,提示其表达可以作为预测胃癌侵袭转移的潜在指标。     

钛表面微沟槽形貌对人牙龈纤维细胞黏着斑蛋白表达的影响

目的 比较不同尺寸微沟槽表面对人牙龈成纤维细胞（HGF）黏着斑蛋白vinculin表达的影响,作为选择种植体穿龈部分微沟槽尺寸的依据之一。方法针对HGF大小,设计不同沟槽尺寸,利用光刻技术制作微沟槽形貌,沟槽宽度和间隔分别为15 μm、30 μm和60 μm,沟槽深度为5 μm和10 μm,分组为T15/5、T15/10、T30/5、T30/10、T60/5和T60/10,光滑钛(T0)表面为对照组。通过免疫荧光、实时荧光定量PCR和蛋白免疫印迹比较vinculin基因和蛋白水平的表达差异。采用SPSS17.0软件包对数据进行统计学分析。结果免疫荧光显示,随着沟槽宽度增加,vinculin 的绿色荧光点增多,mRNA和蛋白水平表达T60最高,T15最低。相同沟槽宽度下,深度改变对该蛋白的表达无显著影响。结论微沟槽形貌有利于vinculin蛋白的表达,表达量随着沟槽的宽度增加而增加。就vinculin蛋白的表达而言,T60组沟槽更适合作为穿龈部分的沟槽尺寸。