受体介导内吞引起的巨噬细胞胞质酸化和胞吐作用

本文利用激光扫描共聚焦显微镜A-CAS570从细胞形态学和功能两方面,研究了刀豆素A(Concanavalin A,Con A)、麦芽凝集素(Wheat Germ Agglutinin,WGA)、酵母多糖(Zymosan A,Z.A)对小鼠腹腔巨噬细胞胞质pH和溶酶体内荧光探针FITC—Dextran排出细胞的影响。结果显示三种配体加入细胞外液10min内,胞质pH很快下降,此后维持在该水平;在15min左右细胞外FITC一Dextran迅速增加,20min后变化趋于停止;在三种配体加入后15min左右,细胞内溶酶体在质膜内侧增多;25—30min溶酶体重新向细胞中央运动。根据上述实验结果,我们认为溶酶体pH升高是触发溶酶体内荧光探针通过胞吐作用排出细胞的必要条件,胞质酸化抑制溶酶体内容物通过胞吐作用排出细胞。配体刺激引起的溶酶体内容物通过胞吐作用排出细胞和胞质酸化是细胞自我调节和保护的一种反映。     

受体介导内吞对巨噬细胞膜电位、胞浆和溶酶体pH的影响

本文利用荧光标记方法测定了刀豆素Ａ、麦芽凝集素、酵母多糖刺激引起的巨噬细胞膜电位、胞浆ｐＨ溶酶体ｐＨ的变化。结果显示三种配体均导致细胞膜电位超极化,胞浆ｐＨ降低、溶酶体ｐＨ或高,三个生理参量趋于稳定时间稍有不同。胞浆ｐＨ的降低可能有抑制内吞的作用,溶酶体ｐＨ上升是触发溶酶体内容物外排的基本因素。内吞引起的这些变化是细胞代谢过程中自我调节和保护的表现。     

巨噬细胞细胞内吞过程中膜受体流动性的测量——两种标记受体…

本文首次实现了细胞内吞过程中膜受体流动性的测量。实验选择巨噬细胞膜和伴刀豆凝集素Ａ（ＣｏｎＡ）。分别用ＣｏｎＡ－Ｂｉｏｔｉｎ＋Ａｖｉｄｉｎ－ＦＩＴＣ（ＡＢＣ法）和ＣｏｎＡ－ＦＩＴＣ（直接法）两种方法标记巨哓细胞膜ＣｏｎＡ受体，比较了这两咱方法标记的巨噬细胞ＣｏｎＡ受体的荧光强度，利用ＦＲＡＰ技术。分别用两种标记方法测量了巨细胞ＣｏｎＡ受体的流动性。结果显示ＣｏｎＡ－Ｂｉｏｔｉｎ＋Ａｖｉｄｉｎ－ＦＩ     

测量细胞内吞过程中膜受体流动性的新方法

本文首次提出用ＡＢＣ（ＡｖｉｄｉｎＢｉｏｔｉｎＣｏｍｐｌｅｘ）法标记细胞膜受体,通过ＦＲＡＰ（ＦｌｕｏｒｅｓｃｅｎｃｅＲｅｃｏｖｅｒｙＡｆｔｅｒＰｈｏｔｏｂｌｅａｃｈｉｎｇ）技术,测量细胞内吞过程中受体流动性变化的方法。实验选择巨噬细胞ＣｏｎＡ受体,比较了用ＣｏｎＡ－Ｂｉｏｔｉｎ＋Ａｖｉｄｉｎ－ＦＩＴＣ（ＡＢＣ法）和ＣｏｎＡ－ＦＩＴＣ（直接法）标记的膜表面ＣｏｎＡ受体荧光强度和内吞过程中受体的流动性测量结果。结果显示ＣｏｎＡ－Ｂｉｏｔｉｎ＋Ａｖｉｄｉｎ－ＦＩＴＣ标记的巨噬细胞膜受体的平均荧光强度比用ＣｏｎＡ－ＦＩＴＣ标记的平均荧光强度高大约３倍;ＡＢＣ法标记的受体,测量结果误差小、灵敏度高;ＣｏｎＡ刺激１５ｍｉｎ后,巨噬细胞膜表面ＣｏｎＡ受体的扩散系数和荧光恢复率较静息状态时明显下降。讨论了两种标记方法对测量结果的影响     

LDL受体介导的血浆低密度脂蛋白胆固醇的内吞

胆固醇是动物细胞细胞膜的重要组成成分,其做为细胞和环境之间的屏障调节细胞膜的流动性。胆固醇是体内所有的类固醇激素和胆酸合成的前体物质,参与体内代谢。同时胆固醇在神经系统的发育中也起着重要的作用。在血浆中胆固醇以低密度脂蛋白和高密度脂蛋白这两种胆固醇运载血脂蛋白的形式运输。动物细胞通过细胞表面的低密度脂蛋白受体(LDL receptor,LDLR)介导的内吞可以从血液中摄取富含胆固醇的低密度脂蛋白,当细胞表面的LDLR的功能缺陷时,可以导致高胆固醇血症,继而引起动脉粥样硬化、冠心病和中风等严重疾病。本文综述了LDL受体的概述及其通过内吞调节血液中低密度脂蛋白胆固醇水平的作用,并对LDL受体的调节进行了阐述。     

巨噬细胞细胞内吞过程中膜受体流动性的测量——两种标记受体方法的比较

本文首次实现了细胞内吞过程中膜受体流动性的测量。实验选择巨噬细胞膜和伴刀豆凝集素A(ConA),分别用Con A-Biotin Avi-din-FITC(ABC法)和Con A-FITC(直接法)两种方法标记巨噬细胞膜Con A受体,比较了这两种方法标记的巨噬细胞Con A受体的荧光强度;利用FRAP(Fluorescence RecoveryAfter Rhotobleaching)技术,分别用两种标记方法测量了巨噬细胞Con A受体的流动性。结果显示Con A-Biotin Avidin-FITC标记的巨噬细胞受体的平均荧光强度比用Con A-FITC标记的平均荧光强度高大约3倍;直接标记法应用于细胞内吞过程中受体流动性的测量在方法学上存在着很大的缺陷,ABC标记法适合于测量细胞内吞过程中膜表面受体的流动性的变化,且灵敏度高、误差小;ABC方法标记受体的测量结果显示,Con A刺激后巨噬细胞膜表面Con A受体的扩散系数和荧光恢复率与静息状态相比呈下降趋势。     

uPA与其抑制剂复合物的受体介导内吞及其应用

尿激酶型纤溶酶原激活物 (ｕＰＡ)是参与细胞外基质降解的重要成分 ,在肿瘤细胞的侵袭和转移中起着重要作用。人们对ｕＰＡ的结构、功能以及它与纤溶酶原激活抑制物 1 (ＰＡＩ 1 )、ｕＰＡ受体 (ｕＰＡＲ)的相互作用都进行了深入的研究。单链ｕＰＡ是一种糖蛋白 ,含有 41 1个氨基酸。其结构可分为四部分 ,依次为 :上皮生长因子区、环状结构区、连接区和丝氨酸蛋白酶区。纤溶酶可裂解Ｌｙｓ1 5 8 Ｉｌｅ1 5 9之间的肽键 ,使单链ｕＰＡ转变为双链ｕＰＡ。ｕＰＡ与其细胞表面受体结合后激活纤溶酶原 ,自身激活也增强。结合在细胞膜上的ｕＰＡ…     

衣霉素对胰岛素受体结合胰岛素及内吞作用的影响

本文研究了人肝癌细胞ＳＭＭＣ－７７２１的胰岛素受体与＾１２５Ｉ－胰岛素结合的条件,并比较了衣霉素处理和对照细胞的结合动力学和内吞作用。结果表明：４℃和ＰＨ８是研究胰岛素受体与配体结合的较佳条件,当０．１ｕｇ／ｍｌ衣霉素处理１８小时,Ｓｃａｔｓｈａｒｄ作图分析指出,胰岛素受体的结合容量降低,每个细胞上的受体位点数减少。Ｈｉｌｌ作图分析说明,胰岛素和受体的亲和力（胰岛素半饱和浓度和表观解离常数）及结合     

ATP对巨噬细胞内吞和溶酶体pH的影响

ＦＩＴＣ－ｄｅｘｔｒａｎ标记培养的小鼠腹腔巨噬细胞溶酶体,ＣｏｎＡ－ＦＩＴＣ标记细胞内吞。用激光扫描共聚焦显微镜测量伴刀豆球蛋白（ＣｏｎＡ）、ＡＴＰ引起的巨噬细胞溶酶体ｐＨ动态变化和ＡＴＰ对细胞内吞ＣｏｎＡ－ＦＩＴＣ的影响。结果显示ＣｏｎＡ引起巨噬细胞溶酶体ｐＨ迅速增加,６ｍｉｎ左右达到峰值（ｐＨ５．７）;ＡＴＰ刺激３０ｍｉｎ后再加入ＣｏｎＡ,溶酶体ｐＨ无明显变化（ｐＨ４．０）;同时加入ＡＴＰ和ＣｏｎＡ,５ｍｉｎ左右溶酶体ｐＨ降到最低点（ｐＨ４．１）;ＡＴＰ对巨噬细胞内吞ＣｏｎＡ－ＦＩＴＣ有明显的抑制作用。探讨了受体介导内吞与溶酶体ｐＨ的关系。     

转铁蛋白/转铁蛋白受体介导的药物运输

转铁蛋白 转铁蛋白受体介导的铁吸收过程是哺乳动物吸收铁的主要途径。近几十年的研究 ,对此过程有了充分了解。作为靶向配体 ,转铁蛋白可以介导多种金属的运输。最新研究表明转铁蛋白可以与抗癌药物、蛋白质、基因等形成复合物 ,靶向肿瘤、血脑屏障、快速分裂等大量表达转铁蛋白受体的组织。转铁蛋白偶联的药物具有靶向性强、毒副小的优点。综述了转铁蛋白 转铁蛋白受体介导的药物运输的最新研究进展。     

Receptor-mediated endocytosis of tuftsin by macrophage cells

A fluorescent analog of the phagocytosis stimulating peptide tuftsin was prepared by coupling tetramethyl rhodamine isothiocyanate to a C-terminal elongated derivative of tuftsin. This analog, Thr-Lys-Pro-Arg-Gly-Lys(N epsilon-tetramethyl rhodamine)-OH, was used to visualize tuftsin receptors on mice macrophage cells by fluorescent image intensification. Fluorescent labelling was carried out at 37 degrees C, using a concentration of 200 nM and 2 microM of the fluorescent tuftsin derivative. The formation of peptide-receptor clusters and their subsequent internalization, as discerned by image intensification, were rapid processes, 5 min and 5-30 min, respectively. Preincubation of macrophages with tuftsin for various time intervals, followed by quantification of the tuftsin receptor using radiolabelled tuftsin, suggest that tuftsin receptors are initially increased in amount (5-7 min) and subsequently reduced (after 10-15 min) as judged by sites available for tritiated tuftsin. The binding studies are rather complementary to the fluorescence observations and support the assumption that the tuftsin receptor on the membrane of the mice macrophage cell is rapidly mobilized.     

Receptor-mediated endocytosis and exocytosis of transferrin in Concanavalin A-stimulated rat lymphoblasts

Iron-loaded transferrin has been shown to be necessary for the support of cell proliferation in culture. This function depends upon interaction of transferrin with a specific high-affinity cell surface receptor. The present report is directed toward determining the consequences of the interaction of transferrin with this receptor on Concanavalin A-stimulated rat lymphocytes. Three specific questions have been posed: (a) Is transferrin endocytosed following binding to its specific receptor in a temperature-dependent fashion? (b) Following endocytosis, is the carrier protein released from the cell in a structurally and functionally intact form? and (c) Is the cell surface transferrin receptor also endocytosed following ligand binding? The results provide affirmative answers to all questions. Using two independent probes of the cell surface versus intracellular location of transferrin we observed that cell-bound transferrin moved from the cell surface to the inside of the cell and subsequently back to the medium. This process occurred in a temperature-dependent fashion. When cells containing only intracellular transferrin were further incubated at 37°C approximately 80% of cell-bound transferrin was released to the medium. Nearly all of this material retained reactivity with antibody to transferrin. In addition, exocytosed transferrin exhibited qualitatively and quantitatively equivalent binding reactivity with the transferrin receptor and showed identical electophoretic mobility on SDS gel electrophoresis. Finally, using similar methodology to that employed with transferrin itself, we provide evidence that the specific receptor is also endocytosed.     

Receptor-mediated endocytosis and cytoskeleton

Receptor-mediated endocytosis is the most specific pathway for macromolecules and macromolecular complexes generally designated as ligands to enter cells. Upon binding to their transmembrane receptors, the ligands enter endocytic vesicles that fuse with each other giving rise to the so-called early endosomes. The sorting of ligand-receptor complexes internalized in these endosomes depends on their nature: metabolic receptors are recycled back to the plasma membrane, while signaling receptors and their ligands (e.g. receptor tyrosine kinases or receptors associated with tyrosine kinase) are delivered to internal vesicles of the multivesicular late endosomes and finally are degraded after interaction with lysosomes. During these processes, endosomes undergo translocation from the cell periphery to the juxtanuclear region, which is accompanied by multiple fusion, invagination, tabulation, and membrane fission events. This review considers modern concepts of the sorting mechanisms of ligand-receptor complexes, the crosstalk between endosomes, microtubules, and actin, and the role of this crosstalk in endosome maturation.     

Receptor-mediated and bulk-phase endocytosis cause macrophage and cholesterol accumulation in Niemann-Pick C disease

These studies explored the roles of receptor-mediated and bulk-phase endocytosis as well as macrophage infiltration in the accumulation of cholesterol in the mouse with Niemann-Pick type C (NPC) disease. Uptake of LDL-cholesterol varied from 514 microg/day in the liver to zero in the central nervous system. In animals lacking LDL receptors, liver uptake remained about the same (411 microg/day), but more cholesterol was taken up in extrahepatic organs. This uptake was unaffected by the reductive methylation of LDL and consistent with bulk-phase endocytosis. All tissues accumulated cholesterol in mice lacking NPC1 function, but this accumulation was decreased in adrenal, unchanged in liver, and increased in organs like spleen and lung when LDL receptor function was also deleted. Over 56 days, the spleen and lung accumulated amounts of cholesterol greater than predicted, and these organs were heavily infiltrated with macrophages. This accumulation of both cholesterol and macrophages was increased by deleting LDL receptor function. These observations indicate that both receptor-mediated and bulk-phase endocytosis of lipoproteins, as well as macrophage infiltration, contribute to the cholesterol accumulation seen in NPC disease. These macrophages may also play a role in parenchymal cell death in this syndrome.     

Linking exocytosis and endocytosis during phagocytosis

Phagocytosis is used by macrophages, dendritic cells and neutrophils to capture and destroy pathogens and particulate antigens. Although localized assembly of actin filaments is the driving force for particle internalization, exocytosis of intracellular compartments, and in particular endocytic compartments, has been shown recently to be required for the early steps of phagosome formation. Here we report on the different compartments undergoing exocytosis during phagocytosis, with a special focus on late endosomes. We then compare this process with secretion from lysosomes or lysosome-related organelles in specialized cells. Finally, we discuss how some of the molecular mechanisms responsible for lysosome-related organelle secretion could also be implicated in phagosome formation.     

Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte

The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by the C. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. elegans predicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme (receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor.