两种人胃癌细胞M期与间期时相质膜表面Con A受体复合物分布及侧向运动

利用荧光标记和荧光漂白恢复方法研究了两种人胃腺癌细胞M期与间期时梠中,细胞膜表面ConA受体复合物分子的分布与侧向运动.结果表明:MGC80-3细胞M期时相与SGC7901细胞间期时相膜表面ConA受体复合物分布近似,其侧向运动方式呈扩散型;SGC 7901 M期时相细胞膜表面ConA受体复合物的分布与MGC80-3细胞间期时和相基本类似,其侧向运动主要是流动型.凡是受体复合物是流动型运动的细胞.其膜上可动分子的百分比都寓于扩散型运为的细胞,P值小于0.01.     

人胃癌不同周期细胞膜表面ConA受体的分布与侧向运动

本文研究了人胃低分化粘液性腺癌细胞MGC 80-3不同周期时相中ConA受体的分布与侧向运动。MGc 80-3细胞经同步化培养,用F-ConA标记。被标记细胞中G_1、S和G_2期呈不连续的分布,但它们之间又存在显著的差异。M期呈较均匀的强荧光分布(与其它时相细胞比较)。荧光漂白恢复方法测定ConA受体复合物侧向运动表明:各个周期时相之间不仅运动方式不同,而且运动速率也有显著差异。M期与G_1期主要表现出扩散型运动;而S期与G_2期表现为流动型运动。G_1期的扩散系数大干M期的;S期的流动速率大于G_2期的。但可动分子百分比以G_2期最高。这些结果表明了ConA受体的动力学性质。它受到细胞周期的调节。     

人胃癌细胞在丁酸钠的诱导下膜分子运动与膜相关骨架的关系

体外培养的人胃癌细胞,用丁酸钠处理后,显示细胞表面FN及细胞胞质微丝的变化,同时用荧光漂白恢复方法对细胞膜ConA受体复合物侧向扩散运动进行测定。结果表明,经丁酸钠处理后的细胞,胞质微丝及细胞膜表面FN均较对照组增加,而细胞膜分子侧向扩散运动减慢,说明与细胞膜相连的细胞骨架的变化对膜分子侧位运动具有牵制作用。     

在膜蛋白分子侧向运动中细胞骨架的作用

本实验采用体外培养的人胃癌细胞,利用免疫荧光技术和FRAP技术研究细胞骨架和膜表面ConA受体复合物的侧向扩散运动.实验分成5组:①用CB处理细胞5小时;②CB处理5小时后,除去药物,换正常培养液培养1小时;③用秋水仙素处理5小时;④秋水仙素处理细胞5小时后,除去药物,换正常培养液培养2小时;⑤对照组.经CB处理细胞后,胞质微丝减少或消失,但是膜ConA受体复合物的侧向扩散运动增加〔D〕=1.05×10~(-1)cm~2/sec,当除去CB后,微丝重新出现,膜ConA受体复合物的侧向扩散运动又减慢〔D〕=0.81×10~(-1)cm~2/sec,与对照组相似.而无论是用秋水仙素处理细胞,还是撤药后微管收复,都不能看到膜表面ConA受体复合物侧向扩散运动的变化.以上结果表明:影响膜表面ConA受体复合物的侧向扩散运动主要在于微丝的作动.     

用原位电泳技术测定艾氏腹水癌细胞膜的流动性

细胞膜组分(膜蛋白、膜脂等)分子能沿膜进行侧向扩散运动,研究这种运动对了解细胞的功能有重要意义。原位电泳(In Situ Electrophoresis)是近年发展起来的测量细胞膜组分侧向扩散的新技术之一。我们用这种技术研究了小鼠艾氏腹水癌细胞膜ConA(Concanavalin A)受体的侧向扩散,并测得在22℃时,其侧向扩散系数为3.2×10~(-10)cm~2/see。实验结果提示,侧向扩散系数具有较大离散性可能是癌细胞的特征之一。     

三种细胞系(人胚肺与小儿包皮成纤维及人胃癌细胞)膜表面ConA受体的再分布和侧向扩散的研究

利用荧光染色和荧光漂白后恢复FRAP技术观察人胚肺成纤维细胞,小儿包皮成纤维细胞和人胃腺癌细胞膜表面Con A受体复合物的分布及其侧向运动.结果表明:它们的分布呈均匀连续性,极为相似.但它们的侧向运动速率各异.两种成纤维细胞差异较小,而人胃癌细胞的侧向扩散系数显著地不同于两种成纤维细胞.因此把凝集素受体的分布与运动行为表达结合起来分析,能更好地认识细胞在生长、分化或恶变过程中膜性状的差异性与共性.     

三种细胞系(人胚肺与小儿包皮成纤维及人胃癌细胞)膜表面ConA受体的再分布和侧向扩散的研究

利用荧光染色和荧光漂白后恢复FRAP技术观察人胚肺成纤维细胞,小儿包皮成纤维细胞和人胃腺癌细胞膜表面Con A受体复合物的分布及其侧向运动.结果表明:它们的分布呈均匀连续性,极为相似.但它们的侧向运动速率各异.两种成纤维细胞差异较小,而人胃癌细胞的侧向扩散系数显著地不同于两种成纤维细胞.因此把凝集素受体的分布与运动行为表达结合起来分析,能更好地认识细胞在生长、分化或恶变过程中膜性状的差异性与共性.     

cAMP对癌细胞增殖周期的影响和膜表面ConA受体分布相关性

用流式光度计、放射自显影和荧光标记等方法研究了体内艾氏腹水癌(EAC)细胞经cA-MP诱导后,在其增殖过程中细胞周期和细胞膜表面ConA受体复合物分布之间的相关性。结果表明:接种后5—9天实验组S期细胞增加45.3％,同时ConA受体复合物分布呈帽状的比率和LI(~3H-TdR掺入)均大于对照组。但G_2+M期细胞的比率及MI却小于对照组,后者呈断续簇状分布的细胞比率大于实验组。至接种后11天,实验组细胞继续簇状分布的比率急剧增加,达6.18倍。这时S期细胞比率和LI均下降,而G_2+M期细胞反而大于对照组。     

ABC法在膜受体流动性测量中的应用

本文首次把ABC法应用于受体流动性测量中的膜表面受体荧光标记,利用FRAP(Fluorescence Recovery After Photobleaching)技术实现了细胞内吞过程中膜受体流动性变化的测量.实验用Con A—Biotin和Avidin—FITC(ABC法)标记巨噬细胞ConA受体,测量ConA刺激不同时间细胞膜表面受体的荧光强度、扩散系数和荧光恢复率的变化.结果显示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变化,且具有较高的灵敏度高;巨噬细胞受ConA刺激后,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降低.     

伴刀豆球蛋白A与巨噬细胞膜受体结合引起膜蛋白和膜脂分子运动及电荷的变化

本文利用荧光漂白恢复,顺磁共振和细胞电泳等技术研究外源性配体伴刀豆球蛋白A与巨噬细胞膜受体结合后膜蛋白及膜脂分子运动以及细胞表面电荷变化,结果表明,细胞膜表面蛋白分子侧向扩散速度减慢;膜脂分子流动性减慢,烃链有序性增强;细胞电泳速度加快。此等对阐明伴刀豆球蛋白A作为外源信息导致细胞膜分子动力学变化以及电荷改变有重要的生物学意义。     

RARβ在胃癌细胞生长调节中的作用

为探讨 RARβ受体介导全反式视黄酸 ( ATRA)抑制胃癌细胞生长的作用机理 ,用 Northern印迹测定 RARβ m RNA表达水平 ,脂质体介导的转染方法将含有 RARβ基因的表达载体转染MKN- 45细胞并稳定表达 ,MTT和软琼脂集落形成等实验测定细胞生长速率和生长状态 ,氯霉素乙酰转移酶活性 ( CAT)测定视黄酸应答元件βRARE的转录活性以及 AP- 1 ( activator protein- 1 )活性 .RARβ在 ATRA敏感细胞株 MGC80 - 3、BGC- 82 3和 SGC- 790 1中表达 ,而在 ATRA抗性细胞株 MKN- 45中不表达 .当 RARβ基因转染 MKN- 45细胞时 ,细胞变为 ATRA敏感 ,由此导致ATRA抑制 MKN- 45细胞生长和软琼脂集落形成 .ATRA可以加强诱导 MGC80 - 3、BGC- 82 3和SGC- 790 1细胞βRARE的转录活性 ,但对 MKN- 45细胞影响不大 ,不能抑制细胞 AP- 1活性 .当RARβ基因转染 MKN- 45细胞后 ,ATRA则能够诱导细胞 βRARE的转录活性 ,并抑制细胞的 AP-1活性 .RARβ表达与 ATRA抑制胃癌细胞生长密切相关 .ATRA诱导 βRARE转录活性和抑制AP- 1活性可能是其调控胃癌细胞生长的机制之一 .     

巨噬细胞吞噬过程中膜磷脂流动性和受体流动性的关系

本文用ＦＲＡＰ（ｆｌｕｏｒｅｓｃｅｎｃｅｒｅｃｏｖｅｒｙａｆｔｅｒｐｈｏｔｏｂｌｅａｃｈｉｎｇ）技术,测量了静息状态和刀豆素Ａ刺激不同时间后巨噬细胞膜磷脂、ＣｏｎＡ受体扩散系数和荧光恢复率的变化。结果显示ＣｏｎＡ刺激后膜磷脂和ＣｏｎＡ受体的扩散系数和荧光恢复率均较静息状态的巨噬细胞明显降低,磷脂流动性的变化与ＣｏｎＡ受体流动性的变化呈正相关。提示受体介导内吞导致的膜磷脂流动性的降低,可能是由于配体与细胞膜上受体结合形成配体－受体复合体,增加了受体的负荷,使受体的流动性降低,进而使膜磷脂的流动性降低。巨噬细胞内吞过程中膜磷脂和ＣｏｎＡ受体流动性的降低,可能还与ＣｏｎＡ刺激后巨噬细胞胞浆ｐＨ值有关。     

Correlation between movement of concanavalin A membrane receptors and cytolysis. A scanning electron microscopy study

The present study was undertaken to test whether cytolysis induced by Concanavalin A (Con A) requires lateral mobility of membranal lectin receptor sites into caps. Treatment of interphase murine mastocytoma cells with 10(-4) M colchicine promoted cap formation by Con A in about 30% of the cells, followed by cytolysis. Pretreatment of the cells with NaN3, low temperature, or glutaraldehyde decreased the degree of capping and, to the same extent, the degree of cytolysis. The addition of antibodies to cells bound with Con A increased the appearance of capping and cytolysis. A linear relationship with a high correlation coefficient exists between the degree of capping and cytolysis, suggesting that lateral mobility of membrane Con A receptors is required for cytolysis by the lectin. The process of cap formation by Con A up to the stage of cytolysis was followed by scanning electron microscopy.     

Cholesterol modulation of nicotinic acetylcholine receptor surface mobility

Nicotinic acetylcholine receptor (AChR) function and distribution are quite sensitive to cholesterol (Chol) levels in the plasma membrane (reviewed by Barrantes in J Neurochem 103 (suppl 1):72–80, 2007). Here we combined confocal fluorescence recovery after photobleaching (FRAP) and confocal fluorescence correlation spectroscopy (FCS) to examine the mobility of the AChR and its dependence on Chol content at the cell surface of a mammalian cell line. Plasma membrane AChR exhibited limited mobility and only ~55% of the fluorescence was recovered within 10 min after photobleaching. Depletion of membrane Chol by methyl-β-cyclodextrin strongly affected the mobility of the AChR at the plasma membrane; the fraction of mobile AChR fell from 55 to 20% in Chol-depleted cells, whereas Chol enrichment by methyl-β-cyclodextrin-Chol treatment did not reduce receptor mobility at the cell surface. Actin depolymerization caused by latrunculin A partially restored receptor mobility in Chol-depleted cells. In agreement with the FRAP data, scanning FCS experiments showed that the diffusion coefficient of the AChR was about 30% lower upon Chol depletion. Taken together, these results suggest that membrane Chol modulates AChR mobility at the plasma membrane through a Chol-dependent mechanism sensitive to cortical actin.     

罗格列酮抑制人胃腺癌SGC7901增殖机制的研究

目的：探讨在体外不同浓度的过氧化物酶体增殖物激活受体γ（PPARγ）激动剂罗格列酮（ROZ）对人胃癌细胞系SGC7901的生长及细胞周期的影响。方法：采用MTT法比色实验、集落形成实验、电子显微镜,透射电镜,流式细胞仪分别观察不同浓度罗格列酮0.08μmol/L,0.4μmol/L,2μmol/L,10μmol/L,50μmol/L,作用于SGC7901细胞,对细胞增殖,细胞形态和细胞周期的影响。结果：ROZ可抑制SGC7901细胞的生长以及SGC7901细胞集落的形成,并呈现剂量依赖性,其半数抑制浓度（IC50）约为50μmol/L。透射电镜低倍镜以及高倍下可见凋亡细胞。流式细胞仪结果显示,ROZ可抑制SGC7901细胞,引起G0/G1期细胞大量增加,S期细胞减少,且细胞周期停滞于G1期。结论：ROZ具有抗肿瘤作用,能够抑制SGC7901细胞的增殖并诱导凋亡,这种作用与其诱导细胞周期G0/G1期的停滞和诱导凋亡作用有关。因此,ROZ有望成为胃癌治疗的辅助用药亦或治疗药,PPARγ有潜力成为肿瘤治疗的新靶点。     

Surface functions during mitosis. III. Quantitative analysis of ligand- receptor movement into the cleavage furrow: diffusion vs. flow

The surface distribution of concanavalin A (Con A) bound to cell membrane receptors varies dramatically as a function of mitotic phase. The lectin is distributed diffusely on cells labeled and observed between mid-prophase and early anaphase, whereas cells observed in late anaphase or telophase demonstrate a marked accumulation of Con A- receptor complexes over the developing cleavage furrow (Berlin, Oliver, and Walter. 1978. Cell. 15:327-341). In this report, we first use a system based on video intensification fluorescence microscopy to describe the simultaneous changes in cell shape and in lectin-receptor complex topography during progression of single cells through the mitotic cycle. The video analysis establishes that fluorescein succinyl Con A (F-S Con A)-receptor complex redistribution begins coincident with the first appearance of the cleavage furrow and is essentially complete within 2-3 min. This remarkable redistribution of surface fluorescence occurs during only a modest change in cell shape from a sphere to a belted cylinder. It reflects the translocation of complexes and not the accumulation of excess labeled membrane in the cleavage furrow: first, bound fluorescent cholera toxin which faithfully outlines the plasma membrane is not accumulated in the cleavage furrow, and, second, electron microscopy of peroxidase-Con A labeled cells undergoing cleavage shows that there is a high linear density of lectin within the furrow while Con A is virtually eliminated from the poles. The rate of surface movement of F-S Con A was quantitated by photon counting during a repetitive series of laser-excited fluorescence scans across dividing cells. Results were analyzed in terms of two alternative models of movement: a flow model in which complexes moved unidirectionally at constant velocity, and a diffusion model in which complexes could diffuse freely but were trapped at the cleavage furrow. According to these models, the observed rates of accumulation were attainable at either an effective flow velocity of approximately 1 micron/min, or an effective diffusion coefficient of approximately 10(- 9) cm2/s. However, in separate experiments the lectin-receptor diffusion rate measured directly by the method of fluorescence recovery after photobleaching (FRAP) on metaphase cells was only approximately 10(-10) cm2/s. Most importantly, photobleaching experiments during the actual period of F-S Con A accumulation showed that lectin-receptor movement during cleavage occurs unidirectionally. These results rule out diffusion and make a process of oriented flow of ligand-receptor complexes the most likely mechanism for ligand-receptor accumulation in the cleavage furrow.     

Distribution and mobility of lectin receptors on synaptic membranes of identified neurons in the central nervous system

The distribution and mobility of concanavalin A (Con A) and Ricinus communis agglutinin (RCA) receptors (binding sites) on the external surfaces of Purkinje, hippocampal pyramidal, and granule cells and their attached boutons were studied using ferritin-lectin conjugates. Dendritic fields of these cells were isolated by microdissection and gently homogenized. Cell fragments and pre- and postsynaptic membranes were labeled with the ferritin-lectin conjugates at a variety of temperatures, and the distribution of lectin receptors was determined by electron microscopy. Both classes of these lectin receptors were concentrated at nearly all open and partially open postsynaptic junctional membranes of asymmetric-type synapses on all three neuron types. Con A receptors were most concentrated at the junctional membrane region, indicating that the mature neuron has a specialized nonrandom organization of carbohydrates on its outer surface. Lectin receptors located on postsynaptic junctional membranes appeared to be restricted in their mobility compared to similar classes of receptors on extrajunctional membrane regions. Labeling with ferritin-RCA and - Con A at 37 degrees C produced clustering of lectin receptors on nonjunctional surfaces; however, Con A and RCA receptors retained their nonrandom topographic distribution on the postsynaptic junctional surface. The restricted mobility of lectin receptors was an inherent property of the postsynaptic membrane since the presynaptic membrane was absent. It is proposed that structures in the postsynaptic density may be transmembrane-linked to postsynaptic receptors and thereby determine topographic distribution and limit diffusion of specialized synaptic molecules. Speicalized receptor displays may play an important role in the formation and maintenance of specific synaptic contacts.     

Distribution and mobility of concanavalin a receptors on isolated guinea pig epidermal cells at various stages of differentiation

Trypsinized guinea pig epidermal cells were separated by velocity sedimentation at unit gravity. Based on the relationship between cell size and both morphological and functional aspects of differentiation, the cells were classified as lower (a diameter <12.5 μM), middle (a diameter between 12.5 and 15 μM), and upper (a diameter >15 μM) epidermal cells. Fresh cells exposed to rhodaminated concanavalin A (Con A) were sedimented and reacted with fluoresceinated anti-Con A serum to distinguish cell surface Con A from intracellular lectin. Labeling at 4°C resulted in a uniform surface distribution of Con A irrespective of cell size. After a 1-hr incubation of Con A-labeled cells in lectin-free medium at 37°C, lower epidermal cells and approximately half of middle epidermal cells internalized Con A/receptor complexes by endocytosis while lectin remained diffusely on the remainder of middle epidermal cells and upper epidermal cells. By electron microscopy, ferritin-Con A was clustered on surface areas and invaginations of the plasma membrane before being endocytosed. We concluded that the differentiation of epidermal cells was accompanied by progressive decrease in endocytosis and, most probably, mobility of Con A receptors.