激光扫描共聚焦显微镜系统及其在细胞生物学中的应用

激光扫描共聚焦显微镜是近十年发展起来的医学图象分析仪器,现已广泛应用于荧光定量测量、共焦图象分析、三维图象重建、活细胞动力学参数监测和胞间通讯研究等方面。其性能为普通光学显微镜质的飞跃,是电子显微镜的一个补充。本文以美国Ｍｅｒｉｄｉａｎ公司的ＡＣＡＳＵＬＴＩＭＡ３１２为例简要介绍了激光扫描共聚焦显微镜系统的结构、功能和生物学应用前景。     

低氧对肺动脉内皮细胞PDGF—B链释放及平滑肌细胞生长的影响

应用蛋白ｄｏｔｂｌｏｔ技术检测了低氧内皮细胞条件培养液（ＨＥＣＣＭ）和常氧内皮细胞条件培养液（ＮＥＣＣＭ）内ＰＤＧＦ相对含量,并利用［３Ｈ］－ＴｄＲ掺入法和流式细胞术观察了ＨＥＣＣＭ和ＮＥＣＣＭ及加入特异ＰＤＧＦ抗体对肺动脉平滑肌细胞（ＰＡＳＭＣ）生长的影响。结果表明,ＨＥＣＣＭ中的ＰＤＧＦ含量明显高于ＮＥＣＣＭ;ＨＥＣＣＭ能明显增强ＰＡＳＭＣ内ＤＮＡ合成,促进ＰＡＳＭＣ从Ｇｏ／Ｇ１期进入Ｓ期;当预先加入ＰＤＧＦ－Ｂ链抗体时,则会明显地抑制ＨＥＣＣＭ对ＰＡＳＭＣ的ＤＮＡ合成,阻止ＰＡＳＭＣ从Ｇｏ／Ｇ１期进入Ｓ期。结果提示,低氧时ＰＡＳＭＣ增殖与肺动脉内皮细胞分泌释放ＰＤＧＦ增加有关     

野生型p53基因对内皮细胞细胞间粘附分子-1表达的影响

细胞间粘附分子－１（ＩＣＡＭ－１）是介导白细胞与内皮细胞粘附的重要粘附分子．为研究野生型ｐ５３基因对内皮细胞ＩＣＡＭ－１表达的影响,分别采用流式细胞术和ＲＴ－ＰＣＲ／ＨＰＬＣ方法测定ＩＣＡＭ－１蛋白及ｍＲＮＡ水平．静息状态的内皮细胞表面结构性地表达有少量的ＩＣＡＭ－１,在肿瘤坏死因子α（ＴＮＦα,１０～１０００Ｕ／ｍｌ）诱导下,其表达呈剂量依赖性增加．将ｐ５３基因导入内皮细胞,则显著抑制ＴＮＦα诱导的内皮细胞表面ＩＣＡＭ－１的表达．ｐ５３基因的导入对静息状态内皮细胞表面结构性表达的ＩＣＡＭ－１影响较小．ｐ５３基因主要通过降低ＩＣＡＭ－１的ｍＲＮＡ水平而抑制内皮细胞表面ＩＣＡＭ－１的表达,但对蛋白的抑制程度小于对ｍＲＮＡ的抑制程度．提示：ｐ５３基因对内皮细胞ＩＣＡＭ－１表达的影响除转录水平控制外,还存在转录后水平的调控     

人类YAC库PCR三维筛选体系的建立及质量考核

为了在知道某个区域、位点、基因或ＤＮＡ片段的部分信息后,能从ＣＥＰＨＹＡＣ库中筛选出与其对应的ＹＡＣ克隆,为进一步研究奠定基础,需要建立一个筛选体系。本文概述了这一筛选体系的建立过程。随后,用两对与已知基因对应的引物进行了筛选验证工作,证明了这一体系的可用性,同时提出了以后筛选的途径,即首先筛选ＹＡＣ库的ＭｅｇａＹＡＣ部分,并以５块板为一组进行筛选。另外,运用荧光原位杂交技术（ＦＢＨ）,对ＣＥＰＨＹＡＣ库的质量及其第一代人类基因组物理图谱进行了考察。我们取２６个ＹＡＣ克隆进行ＦＩＳＨ定位,结果其中嵌合体１３个,占５０％。定位错误的克隆有６个,占２３％。非嵌合体且定位正确的共９个,占３５％。     

松果腺褪黑激素对大鼠下丘脑视交叉上核AVP VIP肽能神经细胞的影响

本研究选用成年健康雄性Ｗｉｓｔａｒ大鼠建立松果腺切除和松果腺褪黑激素（ＭＬＴ）注射模型,并设置正常、松果腺切除假术、生理盐水注射三种对照组。ＭＬＴ注射时间分别为１０００,１７００,２４００。实验组、对照组动物均存活３０天,于上午９００麻醉、固定、取材、切片、ＡＢＣ免疫组化染色、显微图象分析系统（ＭＩＡＳ）相对定量分析处理,目的在于观察ＭＬＴ对ＳＣＮ不同肽能神经细胞的作用。结果表明：ＭＬＴ对ＳＣＮ内ＡＶＰ样神经细胞无显著作用,而可促使其ＶＩＰ样神经细胞中ＶＩＰ含量增多,且此种效应表现出显著的时间窗现象     

中国革螨二新纪录种（蜱螨亚纲：厉螨科、巨螫螨科）

中国革螨二新纪录种（蜱螨亚纲：厉螨科、巨螫螨科）ＴＷＯＮＥＷＬＹＲＥＣＯＲＤＥＤＳＰＥＣＩＥＳＯＦＧＡＭＡＳＩＮＡＦＲＯＭＣＨＩＮＡ（ＡＣＡＲＩ：ＬＡＥＬＡＰＩＤＡＥ,ＭＡＣＲＯＣＨＥＬＩＤＡＥ）￥ＹＥＲＵＩ－ＹＵ（ＸｉｎｊｉａｎｇＩｎｓｔｉｔｕｔｅ...     

平三刺角蝉的替代学名

平三刺角蝉的替代学名ＮＥＷＮＡＭＥＯＦＴＲＩＣＥＮＴＲＵＳＰＬＡＮＩＣＯＲＮＩＳＹＵＡＮＥＴＣＵＩ（ＨＯＭＯＰＴＥＲＡ：ＭＥＭＢＲＡＣＩＤＡＥ）￥ＹＵＡＮＦｅｎｇＩｎｔｉｔｕｔｅｏｆＥｎｔｏｍｏｌｏｇｙ,ＮｏｒｔｈｗｅｓｔｅｒｎＡｇｒｉｃｕｌｔｕｒａ...     

洪湖生态系统钙的地球化学特征

洪湖生态系统钙的地球化学特征杨汉东（中国科学院测量与地球物理研究所,武汉４３００７７）关键词钙,地球化学,生态系统,湖泊ＴＨＥＧＥＯＣＨＥＭＩＣＡＬＣＨＡＲＡＣＴＥＲＩＳＴＩＣＳＯＦＣＡＬＣＩＵＭＩＮＴＨＥＥＣＯＳＹＳＴＥＭＯＦＬＡＫＥＨＯＮＧＨＵ￥...     

紫外线对大豆胚芽化学发光的影响

紫外线对大豆胚芽化学发光的影响范立通（山西大学分子科学研究所,太原０３００６）ＥＦＦＥＣＴＯＦＵＴＲＡＶＩＯＬＥＴＲＡＹＯＮＴＨＥＣＨＥＭＩＬＵＭ－ＩＮＥＳＣＥＮＣＥＯＦＳＯＹＢＥＡＮＥＭＢＲＹＯＮＩＣＳＨＯＯＴ￥Ｆａｎｌｉ－ｔｏｎｇ（Ｉｎｓｔｉｔｕ...     

钙离子对自发性高血压大鼠（SHR）肠系膜血管床活动的影响

本文以ＳＨＲ为实验组,ＷＫＹ为对照组,以灌流压为血管舒缩的指标,对其离体肠系膜血管床,初步研究了钙离子的稳定作用,实验结果如下：（１）ＳＨＲ与ＷＫＹ相比,对血管活性物质去甲肾上腺素（ＮＡ）和氯化钾（ＫＣＩ）均表现出较高反应性。（２）在ＮＡ（１．７×１０Ｍ）使血管床收缩的基础上,Ｃａ￣（＋＋）（２０ｍＭ）使之明显舒张,ＳＨＲ的舒张程度小干ＷＫＹ约１８．１７％（Ｐ＜０．０５,ｎ＝６）．（３）在ＫＣＩ（８０ｍＭ）使血管床收缩的基础上,Ｃａ＋＋（４０ｍＭ）使之明显舒张,ＳＨＲ的舒张程度小于ＷＫＹ约１５．０７％（Ｆ＜０．０５,ｎ＝６）。     

不同固定条件下细胞与活细胞的原子力显微镜实时观察

用原子力显微镜（atom force microscope,AFM）观察固定细胞的最佳条件并在生理溶液中对活细胞实时观察.用不同固定剂和同一固定剂的不同浓度处理细胞;不加任何固定剂而直接在生理溶液中对细胞进行AFM成像.以戊二醛为固定剂并使用0.5%～1%的浓度固定细胞,后用缓冲溶液漂洗,再对细胞进行成像时可获得质量良好的图像.直接在生理溶液中进行观察,成像质量低于使用固定剂的细胞,但保持了细胞的生活原貌.在用原子力显微镜高分辨率观察生理条件下细胞的特点时,需要在制样与观测系统两方面进行改进.     

Use of anti-fluorophore antibody to achieve high-sensitivity immunolocalizations of transporters and ion channels.

We have discovered that the immunoreactivity of the fluorophore Alexa Fluor 488 survives glutaraldehyde and osmium tetroxide fixation and epoxy resin embedding and etching. We have developed new localization methods that for the first time take advantage of this property. The antigen is localized in cryosections using suitable primary antibody and an Alexa Fluor 488-conjugated secondary antibody. Cryosection fluorescence can be photographed for later correlation with electron microscopy (EM) findings. The sections are then further fixed with glutaraldehyde and OsO4, if desired and flat-embedded in epoxy resin. Semi-thin sections are etched completely with sodium ethoxide, whereas thin sections are partially etched. Alexa Fluor 488 is then localized with rabbit anti-Alexa Fluor 488 and goat anti-rabbit conjugated to Alexa Fluor 488 [light microscopy (LM)] or to colloidal gold (EM). A second antigen may also be localized using Alexa Fluor 568. When used without postfixation, these methods produce high-resolution semi-thin, or even thin, sections that retain a high level of fluorescence for LM observations. These methods allow highly sensitive immunolocalizations in tissue while preserving cell fine structure through traditional fixation and epoxy embedding. In demonstration of the methods, we describe the localization of the thiazide-sensitive sodium/chloride cotransporter and the epithelial sodium channel in rat kidney.     

Detection of fragmented DNA in apoptotic cells embedded in LR white: A combined histochemical (LM) and ultrastructural (EM) study.

We developed an improved method for the detection of double-strand DNA breaks in apoptotic cells at both the light (LM) and electron microscopic (EM) levels using a modification of the TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labeling (TUNEL) technique. Cultured rat cerebellar granule cells were exposed to low potassium conditions to induce apoptosis. Twenty-four hr after treatment, one group of cells was fixed in situ with 4% paraformaldehyde and labeled for DNA fragmentation characteristic of apoptosis. Apoptotic cells were visualized with diaminobenzidine (DAB) and viewed by LM. The second group of cells was detached from the culture dish, pelleted, fixed with a 4% paraformaldehyde and 0. 2% glutaraldehyde mixture, and embedded in LR White. For LM, the modified TUNEL technique was performed on 1.5-microm LR White sections and apoptotic cells were visualized using an enzymatic reaction to generate a blue precipitate. For EM, thin sections (94 nm) were processed and DNA fragmentation was identified using modified TUNEL with streptavidin-conjugated gold in conjunction with in-depth ultrastructural detail. Alternate sections of cells embedded in LR White can therefore be used for LM and EM TUNEL-based detection of apoptosis. The present findings suggest that the modified TUNEL technique on LR White semithin and consecutive thin sections has useful application for studying the fundamental mechanism of cell death. (J Histochem Cytochem 47:561-568, 1999)     

Distribution of lectin receptors sites in the zona pellucida of follicular and ovulated rat oocytes.

Carbohydrates of the zona pellucida (ZP) in mammals are believed to have a role in sperm-egg interaction. We have characterized the biochemical nature and distribution of the carbohydrate residues of rat ZP at the light (LM) and electron microscope (EM) levels, using lectins as probes. Immature female rats were induced to superovulate and cumulus-oocyte complexes were isolated from the oviduct, fixed with glutaraldehyde, and embedded in araldite for LM and LR-Gold for EM histochemistry. For examination of follicular oocytes, rat ovaries were fixed with glutaraldehyde and embedded in paraffin. The araldite or paraffin sections were deresined or deparaffinized, respectively, labeled with biotin-tagged lectins as probes, and avidin-biotin-peroxidase complex as visualant. For EM examination, thin LR-Gold sections were labeled with RCA-I colloidal gold complex (RCA/G) and stained with uranyl acetate. LM analyses indicate that in ovulated oocytes the ZP intensely binds peanut agglutinin (PNA); succinylated wheat germ agglutinin, (S-WGA), Griffonia simplisifolia agglutinin-I (GS-I) and soybean agglutinin (SBA), and to a lesser extent, lectins from Ricinus communis (RCA-I), Concanavaia ensiformis (Con A), Ulex europoeus (UEA-I), and wheat germ agglutinin (WGA). The neighboring cumulus cells are considerably less reactive and exhibit membrane staining only with Con A, WGA, and PNA. EM analysis of RCA/G binding revealed intensive binding to the inner layer region of the ZP and moderate binding to cytoplasmic vesicles of the cumulus cells. The ZP of follicular oocytes exhibits a different lectin binding pattern, expressed in staining strongly with PNA and S-WGA, and in a tendency of the lectin receptors to occur in the outer portion of the ZP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Imaging surface of gold-immunolabeled thin sections by atomic force microscopy

Atomic force microscopy (AFM) has been used to image the surface of thin sections of fungal infected plant tissue, with or without post-embedding immunocytochemical labeling with gold conjugates. Plant and fungal cells are easily identified from their size, shape and roughness. The cellular shape is similar to that observed by light or electron microscopy (LM or EM) and some internal organelles can even be individualized. The gold beads are easily observed and counted. Their dimensions varied according to the roughness of the surface, but fit with the expected sizes.     

3D organization and function of the cell: Golgi budding and vesicle biogenesis to docking at the porosome complex

Insights into the three-dimensional (3D) organization and function of intracellular structures at nanometer resolution, holds the key to our understanding of the molecular underpinnings of cellular structure-function. Besides this fundamental understanding of the cell at the molecular level, such insights hold great promise in identifying the disease processes by their altered molecular profiles, and help determine precise therapeutic treatments. To achieve this objective, previous studies have employed electron microscopy (EM) tomography with reasonable success. However, a major hurdle in the use of EM tomography is the tedious procedures involved in fixing, high-pressure freezing, staining, serial sectioning, imaging, and finally compiling the EM images to obtain a 3D profile of sub-cellular structures. In contrast, the resolution limit of EM tomography is several nanometers, as compared to just a single or even sub-nanometer using the atomic force microscope (AFM). Although AFM has been hugely successful in 3D imaging studies at nanometer resolution and in real time involving isolated live cellular and isolated organelles, it has had limited success in similar studies involving 3D imaging at nm resolution of intracellular structure-function in situ. In the current study, using both AFM and EM on aldehyde-fixed and semi-dry mouse pancreatic acinar cells, new insights on a number of intracellular structure-function relationships and interactions were achieved. Golgi complexes, some exhibiting vesicles in the process of budding were observed, and small vesicles were caught in the act of fusing with larger vesicles, possibly representing either secretory vesicle biogenesis or vesicle refilling following discharge, or both. These results demonstrate the power and scope of the combined engagement of EM and AFM imaging of fixed semi-dry cells, capable of providing a wealth of new information on cellular structure-function and interactions.     

海马神经元新连接结构的初步原子力显微观察

目的:利用原子力显微技术(AFM)观察原代培养的海马神经元超微结构及其相互间的连接结构。方法:选择生长良好的海马神经元,用戊二醛固定30min后,固定于AFM基底上进行扫描和观测。结果:正常海马神经元表面光滑,起伏均匀、规律,突起及细胞之间的超微结构清晰,神经元胞体间存在膜性连接及长程非突触性突起连接。结论:神经元间存在直接膜性连接和长程非突触性突起连接结构。     

Microtubule heterogeneity of Ornithogalum umbellatum ovary epidermal cells: non-stable cortical microtubules and stable lipotubuloid microtubules

Lipotubuloids, structures containing lipid bodies and microtubules, are described in ovary epidermal cells of Ornithogalum umbellatum. Microtubules of lipotubuloids can be fixed in electron microscope fixative containing only buffered OsO(4) or in glutaraldehyde with OsO(4) post-fixation, or in a mixture of OsO(4) and glutaraldehyde. None of these substances fixes cortical microtubules of ovary epidermis of this plant which is characterized by dynamic longitudinal growth. However, cortical microtubules can be fixed with cold methanol according immunocytological methods with the use of β-tubulin antibodies and fluorescein. The existence of cortical microtubules has also been evidenced by EM observations solely after the use of taxol, microtubule stabilizer, and fixation in a glutaraldehyde/OsO(4) mixture. These microtubules mostly lie transversely, sometimes obliquely, and rarely parallel to the cell axis. Staining, using Ruthenium Red and silver hexamine, has revealed that lipotubuloid microtubules surface is covered with polysaccharides. The presumption has been made that the presence of a polysaccharide layer enhances the stability of lipotubuloid microtubules.     

Adhesion,phagocytosis and cell surface energy. The binding of fixed human erythrocytes to rat macrophages and polymethylpentene

Fixed human erythrocytes were used as model particles for the study of adhesion and phagocytosis by rat peritoneal macrophages. Erythrocytes were fixed with various concentrations of glutaraldehyde or tannic acid, or were treated with neuraminidase. Adhesion and phagocytosis of these cells were measured. In addition, the surface energy of these erythrocytes and macrophages was estimated by the contact angle technique. Free energies of adhesion, based on the cell surface energies, were correlated with both adhesion and phagocytosis.     

Removal of mucus for ultrastructural observation of the surface of human gastric epithelium using pronase

Background. Helicobacter pylori adhering to the human gastric epithelium causes gastric diseases such as ulcer, carcinoma and lymphoma. It is thus important to observe in detail both the surface of the epithelial cells and the H. pylori that adhered to it for the elucidation of H. pylori‐induced diseases by scanning electron microscopy (SEM). Since the thick mucus layer blocks the observation of the cell surface and the bacteria, it is generally eliminated during the processing for SEM by roughly mechanical methods, but these treatments also demolish the ultrastructure of the cells. We studied the nonmechanical method for removal of mucus layer of gastric epithelium using pronase. Materials and Methods. To determine the optimal concentration of pronase, mucin was used as a substrate for inhibition of the viscosity. Pronase was added in 2% mucin at the concentration of 10, 50, 100, 500, 1000, 2000 or 5000 unit/ml and the flowing time of the mixture was measured. Based on the digestion experiment, biopsied specimens from 24 patients with dyspepsic symptoms were fixed in glutaraldehyde and then washed in rolling with different concentration of pronase. After the pretreatment by pronase, the specimens were treated according to the standard process for SEM. Results. We succeeded in removing the mucus layer on the surface of epithelial cells from the biopsied specimens fixed in glutaraldehyde by rinsing with 2000 unit/ml pronase for 24 hours. Conclusions. Using our digestive method without destroying the ultrastructure, the earliest stage which H. pylori has adhered onto the human gastric epithelium can be observed for the investigation of H. pylori‐induced gastric disorders by SEM.