人心房成纤维细胞体外组织块贴壁培养与鉴定

从建立体外循环将进行心脏手术的风湿性心脏病患者体内无菌下取心脏停跳前右心耳组织，采用组织块贴壁法进行心房成纤维细胞的分离培养，通过光学显微镜观察培养后的细胞形态，并通过免疫细胞化学的方法鉴定所分离培养的细胞。贴壁4—6d后可见长梭形贴壁生长的细胞游离出组织块，细胞生长迅速，2—3d后即呈融合状态，细胞排列紧密，有的交叉重叠生长，胞体较大，细胞浆透明，细胞核较大，呈椭圆形，无自发性搏动。免疫细胞化学检测显示所培养细胞波形蛋白呈阳性反应。通过组织块贴壁法顺利分离人心房成纤维细胞。     

螅状独缩虫(Carchesium polypinum)类中间纤维的初步研究

利用间接免疫荧光和免疫印迹发现螅状独缩虫中存在两种分子质量为58ku和66ku的蛋白。这两种蛋白能分别与抗波形蛋白和抗核纤层蛋白B的抗体反应。分子质量66ku的蛋白位于大核边缘。另外,细胞经分级抽提后大核周围存在核纤层样结构。基于上述结果,可以认为螅状独缩虫中存在类中间纤维。     

眼虫中存在类中间纤维骨架体系

用分级抽提和DGD包埋去包埋技术处理小眼虫(Euglena gracilis),获得了完整的、清晰的细胞纤维网架结构。电镜结果显示纤维的直径在12～14nm,有时呈束状结构,通常以短纤维网络形式存在。免疫印迹证明类中间纤维蛋白的主要成分是一种66kD蛋白。免疫荧光显示类中间纤维蛋白主要分布在各类细胞器的表面,以及细胞表皮的下层。     

胎儿子宫肌层细胞中间丝及收缩蛋白发育

利用免疫组化方法，对１２例胎龄１４～３３周人胎儿子宫肌层细胞波形蛋白、结蛋白及平滑肌肌动蛋白进行研究，结果发现：胎儿子宫肌层平滑肌细胞在发育进程中，首先表达波形蛋白及肌动蛋白，然后表达结蛋白．子宫肌层平滑肌细胞与子宫血管平滑肌细胞存在异质性；胎儿子宫肌层平滑肌细胞同时表达波形蛋白和结蛋白两种中间丝蛋白，是细胞在分过程的特征性表现．成人子宫肌层同时表达波形蛋白及结蛋白，提示成人子宫肌层平滑肌细胞有较强的增殖能力．     

空间诱变致成瘤性减弱的B16细胞株形态学观察

观察动物接种实验致瘤性显著减弱的空间诱变小鼠黑色素瘤B16细胞株的细胞形态、细胞骨架和表面结构与对照细胞株的差异。分别使用激光共聚焦显微镜观察筛选所得阳性细胞株与对照细胞株,以及用荧光素标记的鬼笔环肽染色,观察细胞骨架的异同。应用原子力显微镜(AFM)对筛选所得阳性细胞株和对照细胞株分别成像,观察空间诱变细胞株与对照组细胞株所得图像的差异。激光共聚焦显微镜观察结果显示,从空间诱变细胞株中筛选出的8#细胞株的细胞骨架荧光较对照组增强,细胞骨架纤维比对照组粗大。AFM图像显示,8#细胞株分泌的纤维粘连蛋白的纤维形态和分布与对照组细胞有较大差异,其纤维较为扁平、走行弯曲或呈轻度迂曲状;对照组细胞纤维呈山脊状、放射状,源自细胞伪足部细胞膜。在8#细胞株图像中有少量“孔洞”样结构,而在对照组细胞中未观察到类似结构。     

斑鳜肝脏的组织学及超微结构

光镜观察发现,斑鳜肝脏浆膜极薄,厚度为（1.91±0．89）μm,边缘肝细胞排列紧密,肝小叶极不明显,肝细胞聚集成团,不能分辨出肝细胞索,肝血窦特别丰富,相互交织成网络状,可见枯否氏细胞,无典型汇管区,胆小管非常发达,呈环状或交织成网．电镜观察发现,斑鳜肝细胞具有明显的双态现象,可分为电子密度低的亮细胞和电子密度高的暗细胞．亮细胞多角形或形状不规则,异染色质较少,胞质内粗面型内质网发达,有成层分布现象．暗细胞多角形或卵圆形,异染色质较多,胞质内脂滴特别丰富．两种细胞胞质内线粒体数量众多,可见内含结晶样物质的次级溶酶体,核糖体丰富,糖原分散或聚集成小颗粒状,高尔基复合体靠近狄氏间隙和胆小管区域分布,肝细胞近狄氏间隙和胆小管面由细胞质突起形成丰富微绒毛,说明肝细胞的物质代谢、吸收和分泌功能非常旺盛．     

朝鲜鹌鹑耳蜗的组织学观察

本实验对成体朝鲜鹌鹑耳蜗的组织学结构进行了观察,朝鲜鹌鹑耳蜗由基乳突、听壶和淋巴管三部分组成。听壶由毛细胞、支持细胞和基膜组成;毛细胞呈柱状,支持细胞呈指状。基乳突由毛细胞、支持细胞和基膜组成;毛细胞分成高、中间和矮毛细胞三型。高毛细胞呈柱状,分布在除近端以外各处,在远端全为高毛细胞;中间毛细胞呈壶形和柱形的中间形态,分布在高、矮毛细胞交界处;矮毛细胞呈壶形,分布在除远端以外各处。基乳突的基膜主要由排列疏松的纤维构成。     

生殖期间鳜脑垂体的超微结构观察

对生殖期间鳜脑垂体的超微结构进行观察．脑垂体由神经垂体和腺垂体两部分组成．神经垂体组织中存在2种类型的脑垂体细胞,并可以区分A1、A2和B 3种类型的神经分泌纤维,其终端轴突中具有不同直径和形态的分泌颗粒;B型纤维轴突中含有许多透明小囊泡．腺垂体由前外侧部、中外侧部和中间部构成,分布催乳激素分泌细胞、促肾上腺激素分泌细胞、生长激素分泌细胞、促甲状腺素分泌细胞、促性腺激素分泌细胞、促黑色素激素分泌细胞、PAS-高碘酸-雪夫反应细胞和一种非分泌细胞（星状细胞）类型．本文并讨论了繁殖季节排卵前后期鳜脑垂体的超微结构特点．     

包囊游仆虫细胞的类中间纤维细胞骨架体系

应用生化分级抽提，并结合DGD包埋 去包埋透射电镜样品制备方法显示，包囊游仆虫营养细胞和休眠细胞中，均存在由直径10 nm左右的单根纤维及单根纤维聚集成的纤维束为结构单元形成的类中间纤维细胞骨架体系.其中，营养细胞的类中间纤维构成的细胞质三维网架，在细胞膜内缘以较密集的纤维网占有了整个表质层，在表质层内缘的细胞质深部纤维形成较松散的网络，网内常见附着有细胞器及一些电子密度颗粒；核纤层位于大核核膜内缘，纤维紧密聚集成网；核骨架纤维网分布比较致密，未见有电子密度颗粒附着.休眠细胞中含有与营养细胞相似的纤维网架结构，但位于细胞内不同层次的纤维网比营养细胞中的同种结构要致密得多，这可能与纤毛虫形成包囊时细胞大范围的收缩有关.并且值得注意的是，在休眠细胞包囊壁的内层壁中也观察到相似于中间纤维的纤维网络，其纤维网均匀和致密地分布在整个包囊壁层中.电泳图谱显示，纤毛虫形成包囊后，保留了营养细胞中的部分蛋白条带，失去了部分条带，新产生了一些特异的条带.结果表明，包囊游仆虫的类中间纤维 核骨架体系，是细胞在营养条件下和休眠状态下都稳定存在的结构，它可能起到比微管类骨架更重要的作用.并且休眠细胞中该体系产生的一些特异蛋白条带，可能是纤毛虫休眠生命活动中的重要蛋白.     

视黄酸对胃癌细胞NM—IF系统变化的影响

选择性抽提整装扫描与透射电镜观察显示,人胃腺癌ＭＧｃ８０－３细胞核骨架纤维和中间纤维数量较少、分布不均匀,核纤层为厚薄不一结构,与两类纤维联系不密切．经１０－６ｍｏｌ／ＬＲＡ处理后,细胞核骨架纤维和中间纤维数量增多、结构层次丰富,分布均匀并相互交织成规则网络,两类纤维通过薄层均一的核纤层发生密切联系,形成贯穿整个细胞核质区域的完整体系．表明经ＲＡ诱导处理后ＭＧｃ８０－３细胞的核骨架－中间纤维系统产生了与正常细胞相似的恢复性改变．这种变化是癌细胞恶性表型逆转的重要形态特征和功能表现．     

Redistribution of intermediate filaments during capping of lymphocyte surface molecules

Intermediate filaments (IF) constitute a major cytoplasmic filamentous network of higher eukaryotic cells that is distinct from actin and myosin microfilaments or microtubules. Although structurally similar, these filaments are formed by chemically and antigenically different proteins. Vimentin is the major IF polypeptide of mesenchymal cells and cultured non-mesenchymal cell lines. Recently, we have characterized a monoclonal IgM antibody from a patient with Waldenstr?m's macroglobulinaemia which is directed against vimentin. Using this monoclonal antibody, we have shown by direct immunofluorescence that intermediate filaments of human B and T lymphocytes consist of vimentin. In cells exposed to colcemid, the intermediate filaments retracted into a juxtanuclear aggregate ('coli') characteristic of vimentin filaments. As most components of the cytoskeleton, especially actin and myosin, have been implicated in the capping phenomenon, we investigated the effect of capping of either beta 2-microglobulin or membrane immunoglobulins on the organization of the intermediate filament network. We report that capping of these surface molecules induced the redistribution of vimentin just beneath the cap. When colcemid-treated cells were allowed to cap, the location of the cap always coincided with the coil, suggesting that the anchorage point of intermediate filaments is situated within the uropod.     

NF-L and NF-M are expressed in epithelial cells and coassemble with keratin or vimentin

Neurofilaments (NFs) and cytokeratins are both heteropolymers, which assemble into intermediate filaments (IFs) only when other proper IF subunit proteins are expressed simultaneously. To study the assembly property of NFs, we constructed two recombinant adenovirus which could express NF-L or NF-M, fused with green fluorescent protein (GFP) respectively. Then they were introduced into vero cells, and expressed fusion protein. Double labels of GFP fluorescence and immunofluorescence staining indicated that NF-L-GFP or GFP-NF-M not only coassembled with endogenous vimentins, but also coassembled with keratins into a cytoplasmic network of filaments.     

Site-specific phosphorylation induces disassembly of vimentin filaments in vitro

Intermediate filaments are a major component of the cytoskeleton of eukaryotic cells. Although there appear to be at least five distinct classes of these filaments, cells of mesenchymal origin and most cells in culture contain the intermediate filament composed of the subunit protein vimentin. Vimentin exists in a nonphosphorylated as well as in a phosphorylated form, and there is increased phosphorylation of this protein when the filament undergoes marked redistribution in various cells. The role of phosphorylation on assembly-disassembly and organization of the vimentin filament has remained obscure. We report here a stable and purified system allowing biochemical examination of vimentin filament assembly and disassembly. Using this in vitro system, we carried out stoichiometrical phosphorylations, using purified protein kinases. We obtained evidence for site-specific, phosphorylation-dependent disassembly of the vimentin filament.     

Co-expression of vimentin and cytokeratins in parietal endoderm cells of early mouse embryo

Of the five classes of intermediate filaments found in vertebrate tissues, the cytokeratins are considered unique to epithelial tissues, while vimentin is expressed by endothelial and mesenchymal cells. In neither case is the precise function of the filament system known. Epithelial cells in culture often express vimentin as well as cytokeratins, but co-expression in vivo, as reported for pleomorphic adenomas of the parotid gland and metastatic carcinoma cells in ascites or pleural fluid, is still controversial. Here we report the co-expression of cytokeratins and vimentin in situ, in the parietal endoderm of the mouse embryo 8.5-13.5 days old. This population of individual, motile cells seems to be derived from a conventional epithelium by migration and differentiation. Our results support the idea that vimentin expression is specifically related to reduced cell-to-cell contact, and to the independent existence of a cell following detachment from an epithelial sheet.     

Coalignment of vimentin intermediate filaments with microtubules depends on kinesin.

Intermediate filaments in most types of cultured cells coalign with microtubules. Depolymerization of microtubules results in collapse of vimentin and desmin intermediate filaments to the nucleus where they form a perinuclear cap. Collapse can also be induced by microinjection of antibodies against intermediate filament or microtubule proteins. Thus, two filament systems interact with each other. But the molecules mediating this interaction are unknown. One of the candidates for this role is a microtubule motor kinesin. Recent data showed that kinesin is involved in the plus end-directed movement of the membranous organelles along microtubules such as radial extension of lysosomes in macrophages and centrifugal movement of pigment in melanophores. Here we report that injection of the anti-kinesin antibody into human fibroblasts results in the redistribution of intermediate filaments to a tight perinuclear aggregate but had no effect on the distribution of microtubules. Thus, kinesin is involved not only in organelle movement but also in interaction of the two major cytoskeletal systems, intermediate filaments and microtubules.     

Cytochalasin D does not produce net depolymerization of actin filaments in HEp-2 cells

The altered morphology, disappearance or 'disruption' of actin filaments (microfilaments) in cells treated with cytochalasin has sometimes been attributed to depolymerization of filamentous actin (F-actin) to its globular subunit (G-actin), but attempts to confirm that mechanism have been inconclusive. Treatment of purified actin filaments with cytochalasin B (CB) decreased their viscosity, consistent with depolymerization, which was not, however, revealed by electron microscopy, although the filaments appeared abnormal. CB also increased the ATP-ase activity of F-actin, suggesting that it had been destabilized, while actin filaments in the acrosomal process were not depolymerized. CB or cytochalasin D (CD) can dissolve actin gels (reviewed in ref. 7, see also refs 8 and 9) without depolymerizing their filaments. The 'disrupted' actin structures in CD-treated cells bound heavy meromysin, indicating that at least some of the cellular actin was filamentous. Using a rapid assay for G- and F-actin in cell extracts, based on the inhibition of DNase I, we have found that neither short-nor long-term exposure of HEp-2 cells to CD produce net depolymerization of actin filaments.     

Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins

Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.