单克隆抗体MGd1 对胃癌SGC-7901 细胞增殖和凋亡的影响

目的:研究不同浓度的MGd1对体外培养胃癌细胞SGC-7901增殖及凋亡的影响。方法:采用MTT法测定不同浓度MGd1对SGC-7901生长抑制作用;流式细胞术(FCM)进行细胞凋亡分析。激光共聚焦显微镜观察MGd1抗原(MGd1-Ag)的亚细胞定位。结果:MTT结果显示不同浓度的MGd1均对SGC-7901细胞产生明显的抑制效应(P=0.02);流式细胞术分析发现MGd1可诱导SGC-7901发生凋亡并呈浓度和时间依赖性(P<0.01);共聚焦显微镜结果显示MGd1-Ag主要定位于细胞膜上。结论:以上结果证实胃癌特异性单抗MGd1可抑制SGC-7901的增殖并促进凋亡发生。它可能通过与细胞膜上抗原特异性结合,影响下游信号传导,从而发挥抑制效应。     

胃癌细胞TRAIL受体的表达与其对TRAIL敏感性的关系

目的:探讨胃癌细胞表面TRAIL受体表达水平及其与TRAIL敏感性的关系.方法:PI染色、流式细胞仪检测TRAIL诱导BGC-823及SGC-7901细胞的凋亡率,流式细胞仪检测细胞膜表面四种TRAIL受体-R1、R2、R3、R4的表达情况.结果:TRAIL诱导胃癌细胞凋亡具有剂量和时间依赖性,BGC-823较SGC-7901对TRAIL诱导的凋亡更敏感,TRAIL(100μg·L-1)作用24h的细胞凋亡率分别是59.9%、24.3%.死亡受体TRAIL-R1/DR4、TRAIL-R2/DR5在BGC-823细胞膜表面表达的阳性率高达97.87%和99.42%,而在SGC-7901分别为7.03%和95-31%,诱骗受体TRAIL-R3/DcR1、TRAIL-R4/DcR2在两株细胞膜表面极少表达.结论:胃癌细胞对TRAIL诱导凋亡的敏感性差异可能与细胞膜表面死亡受体有关,尤其与DR4的表达有关.     

单克隆抗体MGd1对胃癌SGC-7901细胞增殖和凋亡的影响

目的：研究不同浓度的MGd1对体外培养胃癌细胞SGC-7901增殖及凋亡的影响。方法：采用MTT法测定不同浓度MGd1对SGC-7901生长抑制作用;流式细胞术（FCM）进行细胞凋亡分析。激光共聚焦显微镜观察MGd1抗原（MGd1-Ag）的亚细胞定位。结果：MTT结果显示不同浓度的MGd1均对SGC-7901细胞产生明显的抑制效应（P=0.02）;流式细胞术分析发现MGd1可诱导SGC-7901发生凋亡并呈浓度和时间依赖性（P〈0.01）;共聚焦显微镜结果显示MGd1-Ag主要定位于细胞膜上。结论：以上结果证实胃癌特异性单抗MGd1可抑制SGC-7901的增殖并促进凋亡发生。它可能通过与细胞膜上抗原特异性结合,影响下游信号传导,从而发挥抑制效应。     

牛精细胞表面凝集素受体的研究

外源凝集素能与细胞表面相应的受体特异性的结合,并导致细胞凝集,因而外源凝集素可用作细胞膜结构的探针,用来研究细胞膜的结构与功能。近年应用外源凝集素对精细胞膜上糖蛋白和凝集素受体的数目与功能状态的研究已取得很大进展,采用异硫氰基荧光素(FITC)或辣根过氧化物酶等标记凝集素来测定膜上凝集素受体的定位与分布,研究认为精细胞膜上受体与受精作用有密切关系。本文报道用具有血型专一性或无血型专一性或FITC标记的凝集素研究牛精细胞膜上凝集素受体的分布结果。     

人胚胎干细胞膜的特异多肽受体研究

目的:通过多肽筛选和比较分析,找到针对人胚胎干细胞(hESC)特异结合的多肽的膜受体蛋白,为相关通路或特异膜表面蛋白下游的研究奠定基础。方法:首先,在前期运用噬菌体展示技术的基础上进行ELISA重筛选,通过对比结合强度的大小,挑选出特异性结合人胚胎干细胞的噬菌体多肽并且进行测序和合成有poly-his标签的多肽;然后运用His Pull-Down系统获得特异结合人胚胎干细胞的某一特殊噬菌体12肽的膜上靶分子受体蛋白;最后质谱测序后通过Mascot数据库和NCBI进行序列信息分析。结果:1通过ELISA重筛选,得到了高特异性结合人胚胎干细胞的两个噬菌体序列,其序列分别为HGAAWGTRTGHV(HGA)和VPATETAQAGHA(VPA)。2通过His Pull-Down实验得到了一个针对多肽VPA的特异性膜蛋白受体。3通过MALD质谱分析以及NCBI的数据库搜索分析,进一步确认这一VPA多肽特异性结合的潜在受体蛋白可能属于HECT超级家族。结论:寻找到一个潜在未知的人胚胎干细胞的特异表面标志物,此膜受体可与VPA多肽特异性结合,为人胚胎干细胞的筛选和鉴定提供了重要指标。     

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.     

c-FLIP在DATS诱导入胃癌SGC-7901细胞凋亡中的作用

目的:探讨二烯丙基三硫(diallyl trisulfide,DATS)诱导入胃癌SGC-7901细胞凋亡及凋亡过程中c-FLIP的变化及意义.方法:采用MTr,westem-blot和细胞免疫组化分别检测DATS对SGC-7901细胞的增殖抑制率及c-FLIP的表达情况.光学显微镜现察凋亡形态,流式细胞术检测凋亡率.结果:MTT结果显示,不同浓度DATS(6,8,10,12,14,16mg.L~(-1)DATS处理SGC-7901细胞24,48小时后,生长抑制率分别为20.4%-79%和36%-90%,DATS抑制作用随浓度及时间逐渐增强(P<0.05).细胞免疫组化和western-blot显示:9.5mg..L~(-1)DATS处理SGC-7901细胞24,48小时后,与对照组相比,c-FLIP的表达下调(P<0.05).光学显微镜:通过9.5mg..L~(-1)DATS作用后24,48小时后,胃癌细胞出现了凋亡形态学改变.流式细胞术检测:经过9.5mg..L~(-1)DATS处理SGC-7901细胞24,48小时后,细胞凋亡率逐渐升高.结论:DATS促进SGC-7901细胞凋亡的机制可能与抑制c-FLIP蛋白的表达有关.     

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.     

白花蛇舌草诱导胃癌SGC-7901细胞凋亡

本研究以人胃癌SGC-7901细胞为实验对象,探讨白花蛇舌草对人胃癌SGC-7901细胞凋亡的影响。使用台盼蓝拒染法检测人胃癌SGC-7901细胞活性,通过普通光学显微镜、荧光显微镜和共聚焦显微镜,观察细胞的形态结构变化,DNA laddy检测DNA片段,Western blotting检测bax和Bcl-2基因的表达,流式细胞术检测细胞周期及线粒体膜电位。结果显示,在一定浓度范围内白花蛇舌草能抑制人胃癌SGC-7901细胞增殖,呈时间和剂量依赖性。24 h、48 h和72 h的半数抑制浓度(IC50)分别为135.886μg/m L,45.84μg/m L和42.56μg/m L。显微镜下观察到细胞皱缩、核裂变、凋亡小体、细胞核裂解、染色质形态改变等现象,DNA出现片段化,细胞阻滞于G1期,Bax蛋白表达上调、Bcl-2的蛋白表达下调。上述表明白花蛇舌草能抑制人胃癌SGC-7901细胞增殖并诱导其凋亡。     

全内角反射技术与转盘式共聚焦技术在细胞膜表面的成像比较

转盘式共聚焦成像是一种高速、高分辨率成像技术,可以在高时间分辨率和空间分辨率的水平观察固定细胞内目标蛋白的分布及活细胞内目标蛋白的动态变化。全内角反射成像是一种观察距离玻片表面某个限定区域内蛋白质的分布和变化的成像技术,常用于观察固定细胞以及活细胞表面的亚细胞结构。该文以中性粒细胞和神经胶质瘤细胞作为观察对象,通过观测固定细胞膜表面蛋白质的分布以及追踪膜标记活细胞的动态变化对两种成像方法进行了比较。结果发现,就目前技术水平而言,二者均可以采集到清晰的细胞边缘,但全内角反射可以拍摄到更清晰的细胞膜表面结构,它在动态拍摄过程中光漂白相对较低,在快速捕捉过程中能够更加全面的捕捉到一个完整的运动过程。     

量子点的近场激发荧光特性研究

近场光学显微镜具有nm量级的空间分辨率,量子点(quantum dots,QDs)荧光探针具有激发谱宽、发射谱线窄、荧光强度高、抗光漂白和稳定性高等优点,两者结合用于生物大分子的成像探测和识别具有广泛的应用前景。用近场光学显微镜对链霉亲和素偶联的QDs进行近场荧光激发,并对其荧光发射特性和光稳定性进行研究,结果表明:近场光学显微镜nm量级的空间分辨率,可以同时观察到了QDs的单体、二聚体和三聚体;QDs的荧光发射强度高,近场荧光像对比度好,单量子点的荧光半高宽达到25nm;对一定入射波长的单色激发光,QDs的近场荧光强度随着激发功率密度的增加线性增加,并很快趋于稳定。与传统的荧光染料如异硫氰酸荧光素相比,QDs的稳定性非常好,在激发功率密度为300W/cm2的近场辐射下,量子点的荧光强度超过6h基本保持不变,其抗光漂白能力远远高于普通荧光染料。     

High-resolution near-field optical imaging of single nuclear pore complexes under physiological conditions

Scanning near-field optical microscopy (SNOM) circumvents the diffraction limit of conventional light microscopy and is able to achieve optical resolutions substantially below 100 nm. However, in the field of cell biology SNOM has been rarely applied, probably because previous techniques for sample-distance control are less sensitive in liquid than in air. Recently we developed a distance control based on a tuning fork in tapping mode, which is also well-suited for imaging in solution. Here we show that this approach can be used to visualize single membrane protein complexes kept in physiological media throughout. Nuclear envelopes were isolated from Xenopus laevis oocytes at conditions shown recently to conserve the transport functions of the nuclear pore complex (NPC). Isolated nuclear envelopes were fluorescently labeled by antibodies against specific proteins of the NPC (NUP153 and p62) and imaged at a resolution of approximately 60 nm. The lateral distribution of epitopes within the supramolecular NPC could be inferred from an analysis of the intensity distribution of the fluorescence spots. The different number densities of p62- and NUP153-labeled NPCs are determined and discussed. Thus we show that SNOM opens up new possibilities for directly visualizing the transport of single particles through single NPCs and other transporters.     

Near-field optical imaging of abasic sites on a single DNA molecule

Scanning near-field optical microscopy (SNOM) imaging was performed to allow for the direct visualization of damaged sites on individual DNA molecules to a scale of a few tens of nanometers. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labelled with a biotinlylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. By optimizing the performance of the SNOM technique, we could obtain high contrast near-field optical images that enabled high-resolution near-field fluorescence imaging using optical fiber probes with small aperture sizes. High-resolution near-field fluorescence imaging demonstrated that two abasic sites within a distance of 120 nm are clearly obtainable, something which is not possible using conventional fluorescence in situ hybridization combined with far-field fluorescence microscopy.     

High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies

BackgroundHigh-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy.MethodsScanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever.ResultsIn tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated.ConclusionsThis study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research.General significanceWe achieved an imaging rate of ~3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.     

近场扫描光学成像结合量子点标记的纳米技术在细胞生物学中的应用

近场扫描光学显微镜（NSOM）对传统的光学分辨极限产生了革命性的突破,可在超高光学分辨率下无侵人性和无破坏性地对生物样品进行观测。量子点（QDs）具有极好的光学性能,如荧光寿命长、激发谱宽、生物相容性强、光稳定性好等优点,适合先进的生物成像。NSOM结合QDs标记的纳米技术被应用在细胞生物学中。通过纳米量级NSOM免疫荧光成像（50nm）对特定蛋白分子在细胞表面的动态分布进行可视化研究和数量化分析,阐明了蛋白分子在不同细胞过程中的作用机制。因此,NSOM／QD基成像系统提供了单个蛋白分子最高分辨率的荧光图像,为可视化研究蛋白分子机制的提供了一种强有力的工具。     

Scanning Near-field Optical/Atomic Force Microscopy detection of fluorescence in situ hybridization signals beyond the optical limit

Fluorescence in situ hybridization (FISH) is widely used in molecular biological study. However, high-resolution analysis of fluorescent signals is theoretically limited by the 300-nm resolution optical limit of light microscopy. As an alternative to detection by light microscopy, we used Scanning Near-field Optical/Atomic Force Microscopy (SNOM/AFM), which can simultaneously obtain topographic and fluorescent images with nanometer-scale resolution. In this study, we demonstrated high-resolution SNOM/AFM imaging of barley chromosome (Hordeum vulgare, cv. Minorimugi) FISH signals using telomeric DNA probes. Besides detecting the granular structures on chromosomes in a topographic image, we clearly detected fluorescent signals in telomeric regions with low-magnification imaging. The high-resolution analysis suggested that one of the telomeric signals could be observed by expanded imaging as two fluorescent regions separated by approximately 250 nm. This result indicated that the fluorescent signals beyond the optical limit were detected with higher resolution scanning by SNOM/AFM.     

Nano-probing of the membrane dynamics of rat pheochromocytoma by near-field optics

High-resolution analysis of activities of live cells is limited by the use of non-invasive methods. Apparatuses such as SEM, STM or AFM are not practicable because the necessary treatment or the harsh contact with system probe will disturb or destroy the cell. Optical methods are purely non-invasive, but they are usually diffraction limited and then their resolution is limited to approximately 1 microm. To overcome these restrictions, we introduce here the study of membrane activity of a live cell sample using a Scanning Near-field Optical Microscope (SNOM). A near field optical microscope is able to detect tiny vertical movement on the cell membrane in the range of only 1 nm or less, about 3 orders of magnitude better than conventional optical microscopes. It is a purely non-invasive, non-contact method, so the natural life activity of the sample is unperturbed. In this report, we demonstrated the nanometer-level resolving ability of our SNOM system analyzing cardiomyocytes samples of which membrane movement is known, and then we present new intriguing data of sharp 40 nm cell membrane sudden events on rat pheochromocytoma cell line PC12. All the measurements are carried out in culture medium with alive and unperturbed samples. We believe that this methodology will open a new approach to investigate live samples. The extreme sensitivity of SNOM allows measurements that are not possible with any other method on live biomaterial paving the way for a broad range of novel studies and applications.     

The lipid raft hypothesis revisited - New insights on raft composition and function from super-resolution fluorescence microscopy

Recently developed super‐resolution microscopy techniques are changing our understanding of lipid rafts and membrane organisation in general. The lipid raft hypothesis postulates that cholesterol can drive the formation of ordered domains within the plasma membrane of cells, which may serve as platforms for cell signalling and membrane trafficking. There is now a wealth of evidence for these domains. However, their study has hitherto been hampered by the resolution limit of optical microscopy, making the definition of their properties problematic and contentious. New microscopy techniques circumvent the resolution limit and, for the first time, allow the fluorescence imaging of structures on length scales below 200 nm. This review describes such techniques, particularly as applied to the study of membrane organisation, synthesising newly emerging facets of lipid raft biology into a state‐of‐the art model. Editor's suggested further reading in BioEssays: Super‐resolution imaging prompts re‐thinking of cell biology mechanisms Abstract and Quantitative analysis of photoactivated localization microscopy (PALM) datasets using pair‐correlation analysis Abstract     

A new filtering technique for removing anti‐Stokes emission background in gated CW‐STED microscopy

Stimulated emission depletion (STED) microscopy is a prominent approach of super‐resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time‐gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub‐diffraction spatial resolution. If the time‐gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW‐STED microscope. However, time‐gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti‐Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti‐Stokes background. The method hinges on the uncorrelated nature of the anti‐Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two‐photon‐excitation with gated CW‐STED microscopy is demonstrated. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Chemically resolved imaging of biological cells and thin films by infrared scanning near-field optical microscopy

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths (lambda) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 micro m ( approximately lambda/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research.