人卵巢癌细胞微观形态的AFM观察

目的:探讨原子力显微镜(AFM)在人卵巢癌细胞微观形貌表征方面的应用。方法:应用原子力显微镜分别观察培养的高低转移的人卵巢癌细胞及其周围纤连蛋白原纤维和经过紫杉醇药物处理后的细胞的微观形态,进一步对正常的卵巢癌细胞组织和经过紫杉醇药物处理后的卵巢癌细胞组织经过超声波处理后组织的微观结构进行AFM成像观察。结果:高转移特性的卵巢癌细胞周围的纤维少而短;而低转移特性的卵巢癌细胞周围的纤维多且长。经过紫杉醇药物处理后的细胞的形态发生了变化,结构呈现不规则状,且与基底没有纤维连接。结论:不同类型的卵巢癌细胞受其生物功能的影响在基底上的形态不同。药物处理后的癌细胞微观组织发生了变化,与其核溶,核碎和核解的生理变化过程相符。正常的卵巢癌细胞组织结构之间的黏附力较小,故超声波处理后呈现分散的分布。经过紫杉醇药物处理后的卵巢癌细胞组织结构之间的黏附力较大,超声波处理后分布在基底上的结构较紧凑。     

识别宿主细胞黏附基质分子的细菌表面组分

许多细菌表面具有能识别并黏附于宿主细胞外基质(如纤连蛋白、胶原、纤维蛋白原等)的表面成分,统称为识别黏附基质分子的微生物表面组分。本文就其性质、结构,以及它们在细菌致病过程中的作用作一综述。     

整合素及其在胚泡植入中的作用

整合素是一类由α、β亚基构成的异二聚体粘附分子,能够与胶原蛋白、纤连蛋白和玻连蛋白等细胞外基质组分相互作用,调节细胞粘附和通讯.作为双向传递分子,整合素通过“胞内→胞外”和“胞外→胞内”两种方式介导细胞信号传递.成功的植入是侵入性的胚泡和接受性的子宫内膜相互作用的结果,整合素能够调节胚泡滋养层与子宫内膜之间的细胞-细胞及细胞-细胞外基质相互作用,是“植入窗口”期子宫内膜接受性的标记分子.     

肌动蛋白的原子力显微镜研究

原子力显微镜 (AFM )是一种能够在生理条件下对生物大分子、活细胞表面以及细胞膜下结构进行在体或离体研究的强有力的新型工具 ,具有原子级的成像分辨率和纳牛顿级的力测定功能。目前原子力显微镜已被广泛地应用于生物大分子、超分子体系的结构解析、动力学过程观察 ,分子力学研究及细胞功能鉴定。原子力显微镜能够通过尖锐探针扫描待测样品表面 ,收集被测样品表面地貌坐标数据从而对单分子或细胞进行成像或操作 ,并能通过移动探针、记录探针与样品之间的作用力 ,对生物大分子 (蛋白质、核酸和多糖等 )的结构力学特性进行分析以获取分子构象、功能及其相互关系的有用信息。肌动蛋白是一种细胞内普遍存在 ,具有广泛、复杂生理功能的重要蛋白质 ,原子力显微镜的各项功能已广泛地用于肌动蛋白结构、功能及动力学研究。通过综述原子力显微镜在肌动蛋白研究中的应用 ,阐明了原子力显微镜在现代生命科学研究中的重要意义及巨大应用前景。     

微原纤维

微原纤维有Ⅵ型胶原蛋白微原纤维和原纤维蛋白微原纤维二种。前者广泛存在于固有结缔组织中,确切功能尚不清楚。原纤维蛋白微原纤维由原纤维蛋白、微原纤维相关蛋白、潜在转化生长因子β结合蛋白等成分构成,主要作为弹性纤维的组成部分,并参与弹性纤维的形成,其次是单独地分布于器官的细胞外基质中,提供一种柔韧性的连接方式。原纤维蛋白的基因突变可引发马凡综合征及相关的微原纤维病。     

兔网织红细胞与K_(562)细胞胞质杂交体(K-RRneo)核基质-中间纤维体系的研究

本文利用脂质体转基因技术与细胞融合相结合的方法所建立的杂交细胞为研究对象,采用选择性多步抽提配合整装电镜及Western 印迹分析等技术,系统观察了兔网织红细胞、人红白血病K_(562)细胞及两者融合形成的胞质杂交体K-RRneo 细胞的核基质-中间纤维体系结构,着重分析比较了它们之间的胞质波形蛋白纤维成分的变化。实验结果表明:K_(562)细胞的中间纤维为放射状分布,核纤层为网层状,兔网织红细胞胞质中间纤维为细网格状,其间存有不规则的致密物。胞质体杂交细胞(K-RRneo)的核纤层结构稀薄,核基质较亲代K_(562)细胞致密,中间纤维构型呈现与网织红细胞相似的网格状。中间纤维蛋白电泳及Western 印迹结果亦显示K-RRneo 细胞与网织红细胞的带型类似,即缺乏聚合型波形蛋白及聚合前体物波形蛋白单体,仅检测到解聚前、后而与细胞其它组分结合的波形蛋白复合物。这一生化和超微结构特征提示,红细胞排核可能与波形蛋白纤维的解聚及Vimentin 基因关闭有关。实验结果为排核前细胞内原已装配好的Vimentin 趋于解聚,引起中间纤维瓦解,造成核偏位、固缩而最终排出细胞外的排核机制提供了证据。     

肌腱蛋白R(Tenascin-R)研究进展

肌腱蛋白R(tenascin-R, TN-R)是一种重要的细胞外基质糖蛋白(extracellular matrix,ECM).分布于中枢神经系统, 主要在髓鞘形成早期的少突胶质细胞中表达,成熟的胶质细胞及某些神经元(如脊髓,视网膜, 小脑和海马的中间神经元)也有表达.TN-R具有复杂的结构,由三种不同的结构域组成,从氨基端到羧基端依次为:类似于表皮生长因子的重复片段, 类似于Ⅲ型纤连蛋白重复片段,类似(血)纤维蛋白原片段组成.TN-R具有多种复杂的功能, 对神经元具有排斥作用,促进或抑制神经元突起的生长, 诱导神经元形态的极性化, 并和髓鞘的形成有关,TN-R结构的复杂性和功能的多样性提示,TN-R有多个受体存在,已经发现的受体有F3/F11,MAG,XL1,Xprocan等.     

N─糖链在细胞粘附中的作用

参与细胞与细胞,细胞与基质相互作用的细胞粘附分子均为含N─糖链的糖蛋白,N─糖链在细胞的识别、粘附过程中起一定的作用。本文就纤连蛋白、层粘连蛋白、整合蛋白、选择蛋白和凝集素受体等分子中N─糖链与细胞识别和粘附关系加以讨论。     

衣霉素对纤连蛋白与HT1080细胞结合的影响

衣霉素对纤连蛋白与ＨＴ１０８０细胞结合的影响曹立环,查锡良（上海医科大学生物化学教研室,２０００３２）关键词纤连蛋白;纤连蛋白受体;衣霉素;Ｎ－糖链配体和受体分子上糖链的功能已成为近几年来复合糖领域研究的新热点之一。以Ｎ－糖链的合成抑制剂阻断或改变Ｎ...     

原子力显微镜对纳米生物结构的观察和操纵

原子力显微镜不仅能对纳米生物结构进行观察,而且也能对其进行操纵。对纳米生物结构的观察已深入到生物大分子结构水平。原子力显微镜对生物大分子的操纵包括从染色质中提取DNA用于基因分析、对膜蛋白的结构进行观察、对蛋白构象进行可控操纵等。这些纳米技术的应用将揭示生物系统更多的结构和功能信息。     

Structural changes in human type I collagen fibrils investigated by force spectroscopy

In the field of biomechanics, collagen fibrils are believed to be robust mechanical structures characterized by a low extensibility. Until very recently, information on the mechanical properties of collagen fibrils could only be derived from ensemble measurements performed on complete tissues such as bone, skin, and tendon. Here, we measure force-elongation/relaxation profiles of single collagen fibrils using atomic force microscopy (AFM)-based force spectroscopy (FS). The elongation profiles show that in vitro-assembled human type I collagen fibrils are characterized by a large extensibility. Numerous discontinuities and a plateau in the force profile indicate major reorganization occurring within the fibrils in the 1.5- to 4.5-nN range. Our study demonstrates that newly assembled collagen fibrils are robust structures with a significant reserve of elasticity that could play a determinant role in the extracellular matrix (ECM) remodeling associated with tissue growth and morphogenesis.     

Characterization of Morphological and Cytoskeletal Changes in MCF10A Breast Epithelial Cells Plated on Laminin-5: Comparison with Breast Cancer Cell Line MCF7

The extracellular matrix regulates functional and morphological differentiation of mammary epithelial cells both in vivo and in culture. The MCF10A human breast epithelial cell line is ideal for studying these processes because it retains many characteristics of normal breast epithelium. We describe a distinct set of morphological changes occurring in MCF10A cells plated on laminin-5, a component of the breast gland basement membrane extracellular matrix. MCF10A cells adhere and spread on laminin-5 about five times more rapidly than on fibronectin or uncoated surfaces. Within 10 minutes from plating on laminin-5, they send out microfilament-rich filopodia and by 30 minutes acquire a cobblestone appearance with microfilaments distributed around the cell periphery. At 90 minutes, with or without serum, >75% of the MCF10A cells plated on laminin-5 remain in this stationary cobblestone phenotype, while the remainder takes on a motile appearance. Even after 18 hours, when the culture is likely entering an exponential growth phase, the majority of cells maintain a stationary cobblestone appearance, though motile cells have proportionally increased. In contrast, the fully transformed, malignant human breast epithelial cells, MCF7, never acquire a stationary cobblestone appearance, do not organize peripheral microfilaments, and throughout the early time points up to 120 min appear to be constantly motile on laminin-5. We propose that changes in morphology and microfilament organization in response to laminin-5 may represent a benchmark for distinguishing normal vs. malignant behavior of epithelial cells derived from the mammary gland. This may lead to better model systems for studying the interactions between breast epithelium and the basement membrane extracellular matrix, which appear to be deregulated in processes like carcinogenesis and metastasis.     

Characterization of morphological and cytoskeletal changes in MCF10A breast epithelial cells plated on laminin-5: comparison with breast cancer cell line MCF7

The extracellular matrix regulates functional and morphological differentiation of mammary epithelial cells both in vivo and in culture. The MCF10A human breast epithelial cell line is ideal for studying these processes because it retains many characteristics of normal breast epithelium. We describe a distinct set of morphological changes occurring in MCF10A cells plated on laminin-5, a component of the breast gland basement membrane extracellular matrix. MCF10A cells adhere and spread on laminin-5 about five times more rapidly than on fibronectin or uncoated surfaces. Within 10 minutes from plating on laminin-5, they send out microfilament-rich filopodia and by 30 minutes acquire a cobblestone appearance with microfilaments distributed around the cell periphery. At 90 minutes, with or without serum, > 75% of the MCF10A cells plated on laminin-5 remain in this stationary cobblestone phenotype, while the remainder takes on a motile appearance. Even after 18 hours, when the culture is likely entering an exponential growth phase, the majority of cells maintain a stationary cobblestone appearance, though motile cells have proportionally increased. In contrast, the fully transformed, malignant human breast epithelial cells, MCF7, never acquire a stationary cobblestone appearance, do not organize peripheral microfilaments, and throughout the early time points up to 120 min appear to be constantly motile on laminin-5. We propose that changes in morphology and microfilament organization in response to laminin-5 may represent a benchmark for distinguishing normal vs. malignant behavior of epithelial cells derived from the mammary gland. This may lead to better model systems for studying the interactions between breast epithelium and the basement membrane extracellular matrix, which appear to be deregulated in processes like carcinogenesis and metastasis.     

Magnetic force microscopy of iron oxide nanoparticles and their cellular uptake

Magnetic force microscopy has the capability to detect magnetic domains from a close distance, which can provide the magnetic force gradient image of the scanned samples and also simultaneously obtain atomic force microscope (AFM) topography image as well as AFM phase image. In this work, we demonstrate the use of magnetic force microscopy together with AFM topography and phase imaging for the characterization of magnetic iron oxide nanoparticles and their cellular uptake behavior with the MCF7 carcinoma breast epithelial cells. This method can provide useful information such as the magnetic responses of nanoparticles, nanoparticle spatial localization, cell morphology, and cell surface domains at the same time for better understanding magnetic nanoparticle‐cell interaction. It would help to design magnetic‐related new imaging, diagnostic and therapeutic methods. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Comparative study of p53 expression in human carcinoma cell lines A549 and MCF7 under anticancer drug treatment

The effect of anticancer drugs on the expression of p53 protein in tumor cells was studied using the Western Blot analysis. Human lung carcinoma cell line A549 and human breast carcinoma cell line MCF7 sensitive (WT) and resistant (DOX/R) to doxorubicin were used. An increase in p53 protein expression was found in A549 and MCF7 (WT) cells treated with cisplatin, methotrexate, and doxorubicin, whereas the level of p53 was not statistically significantly changed in the MCF7 DOX/R cells. In the untreated MCF7 DOX/R cells the level of p53 protein was markedly higher than in the untreated WT MCF7 cells. A potential role of p53 protein in the development of doxorubicin-resistance in carcinoma cells is discussed.     

Micro-heterogenous expression of peanut agglutinin-binding sites in the extracellular matrix of cultured cells

Using double-label techniques with fluorochrome-conjugated peanut agglutinin (PNA) and indirect immunofluorescence with rabbit species-specific anti-fibronectin antibodies and a mouse monoclonal anti-fibronectin, the extracellular matrix (ECM) of cultured human and mouse fibroblasts (Hell7 and 3T3K) and human bladder epithelial cells (T24) was studied. The antibodies and PNA co-localized extensively. However, a small but consistent degree of micro-heterogeneity was revealed insofar as both PNA-positive fibronectin-negative fibrils as well as PNA-negative fibronectin-positive fibrils were observed. Fibronectin production by T24 cells (but not fibroblasts) was influenced by the growth medium, but this did not affect the heterogeneity. Trypsin removed most cell-surface fibronectin and all PNA-binding sites, but did not account for the observed phenomenon. Intracellular fibronectin, whether present naturally or induced to accumulate by culture in presence of Monensin, was PNA-negative. These data suggest that PNA-binding sites appear on fibronectin as a consequence of incorporation into the extracellular matrix, and that the resultant heterogeneity of spatial expression of beta-galactose-like residues may offer a mechanism whereby mesenchymal cells could modulate the behaviour of overlying cell-types.     

Fibronectin organization under and near cells

Polymerization of soluble fibronectin molecules results in fibres that are visible as networks using fluorescently labelled fibronectin protomers or by antibody labelling. Displacement of fibres composed of modified protomers in living cells provides information regarding matrix structure, organization, and movement. A static analysis of fibronectin structures and patterns of organization provide insight into their reorganization during adhesion and motility. Confocal microscopy and atomic force microscopy (AFM) reveal fibronectin-containing networks aligned in arrays perpendicular to the retracting cell edge and in apparently disordered networks of fibres under the cell. The change in patterns suggests a reorganization of fibronectin from disordered arrays used for adhesion into ordered arrays during movement of the cell. Comparison of confocal images with corresponding AFM images confirms that the fibres left on the surface as the cell moves away do contain fibronectin. The orientation of these fibres relative to the tail (uropod) and the receding edges of the cell leads us to propose that cells generate a force on the fibres that exceeds the adhesion force of the fibres to the surface causing them to pull fibronectin fibres into straight arrays. However, when the fibres are parallel to the direction of pull, the fibres remain attached to the surface. The data supports the hypothesis that disorganized, linear fibres are the product of Fn polymerization induced by the cell beneath it and serve to adhere the cell to the substrate as the cell spreads, whereas arrays of fibres found outside the cell are formed as existing fibrils and reorganize during cell motility.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20 h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~ 10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5β1 integrins. However, on vitronectin-adherent cells, α5β1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvβ3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5β1 integrin activation and fibronectin fibril extension.