显微镜技术在植物细胞自噬研究中的应用

细胞自噬是真核生物对细胞内物质进行循环利用的重要途径.在植物中已发现的细胞自噬有两种形式,即微自噬和巨自噬.自噬在植物体的生长发育、免疫应答、胁迫反应、衰老等过程中具有非常重要的作用.显微镜技术是研究细胞自噬的重要手段,与生物化学和分子生物学技术相结合,荧光显微镜,激光扫描共聚焦显微镜,电子显微镜等技术的相互印证,大大地推动了对细胞自噬过程和机理的研究.本文扼要概述了显微镜技术在植物细胞自噬研究中的应用.     

大鼠坐骨神经再生过程中的神经元轴突自噬作用

目的：探讨大鼠坐骨神经变性轴突清除中的自噬作用。方法：横切大鼠坐骨神经制作wallerian变性模型,造模后不同时间点取远断端组织行电镜结构观察和酸性磷酸酶(AcPase)活性检测。结果：坐骨神经横切后轴突发生变性,主要变化为术后第5h～2d轴质肿胀,轴突与髓鞘分离,术后第4d轴质浓缩,轴突与髓鞘完全分离形成游离轴突体。术后初期变性轴突主要形成大小不等的空泡,后期轴突与髓鞘完全分离并形成较大的游离轴突体,轴突体外包一层轴突膜是神经元细胞膜的延续,轴突体轴质浓缩,含大量各级自噬泡和纵横交错的神经丝、微管和微丝。经酸性磷酸酶(AcPase)染色证实自噬泡均呈AcPase阳性,第7d后轴突体被降解吸收,形成的空腔内偶见巨噬细胞。结论：大鼠坐骨神经再生过程中变性轴突的清除主要靠轴突自身的自噬和施万细胞吞噬机制,而巨噬细胞只起辅助作用。     

纳米金棒诱导A549细胞产生自噬的机制研究

纳米金棒(AuNRs)具有众多独特的属性,已广泛运用于生物医学领域,但其是否具有潜在的生物危害尚有争议.作者运用了激光扫描共聚焦显微镜技术、western blotting技术和其他分子生物学方法从细胞氧化应激的角度探讨了AuNRs诱导A549细胞产生自噬的分子机制.研究结果表明,4μg·mL-1的AuNRs处理6 h能够诱导A549细胞自噬标志蛋白LC3-Ⅱ表达增加,LC3蛋白从细胞核转移至细胞质并形成自噬小泡.进一步研究发现,AuNRs能够降低A549细胞线粒体膜电位、ATP含量、UCP2蛋白表达水平以及细胞抗氧化能力并导致活性氧蓄积,后者可能最终引起细胞产生自噬.而10 mmol·L-1抗氧化剂NAC能够逆转上述线粒体及细胞功能的改变,并抑制自噬的发生.这一研究为深入认识其生物危害及可能机制提供了有力的实验证据.     

重力矢量对骨细胞自噬影响的初探

自噬是细胞降解自身成分并重新利用具有多种生理功能的过程,许多原因会导致细胞自噬。模拟微重力会引起细胞自噬增强,但对单一重力矢量对自噬作用的影响研究较少。本研究选择力学敏感骨细胞MLO-Y4,应用激光共聚焦显微镜和透射电镜观察细胞自噬,通过改变细胞培养基底取向方法,比较重力矢量与饥饿对骨细胞自噬作用的影响。结果表明,饥饿可以诱导正置与倒置组细胞发生明显自噬,重力矢量或者说单纯改变培养基底取向并不能诱导MLO-Y4细胞自噬的发生。     

内分泌细胞溶酶体的超微结构研究

用电镜细胞化学方法研究内分泌细胞溶酶体的结构与功能。研究结果表明，所有内分泌细胞都存在丰富的溶酶体，分泌自噬作用普通存在于分泌肽类激素的细胞中，自体吞噬活动跃是分泌类固醇素细胞的重要特点。溶酶体通过分泌自噬和自体吞噬分别在两类内分泌细胞中参与激素分泌的调节。     

自噬在PDT治疗中的作用及其机制研究进展

自噬是真核生物进化过程中高度保守的分解代谢和循环再利用的过程,在维持细胞状态、内环境稳态,尤其是在细胞营养不足、氧化损伤等不利环境时起着重要作用.近来研究发现自噬参与了光动力疗法的作用过程,其机制较为复杂,而且不同程度的自噬对光动力疗法杀伤效应的影响截然不同.光动力疗法是利用特定波长的光照激活光敏剂分子,通过能量转换促...     

人miR-34a靶基因的生物信息学分析及载体构建

目的对人miR-34a(hsa-miR-34a)下游靶基因进行预测,并构建含自噬相关基因靶结合序列的GFP报告基因载体。方法使用TargetScan Release 5.1和RNA22软件分析hsa-miR-34a可能的靶基因,并利用Gominer软件对靶基因功能进行分析。根据预测结果,合成含自噬相关基因hsa-miR-34a靶序列的寡核苷酸,以pEGFPC2为载体构建系列质粒,并检测各载体的真核表达情况。结果预测结果显示hsa-miR-34a共有2904个下游靶基因,功能涵盖生物过程、细胞组分、分子功能三大类,其中与自噬作用直接相关的靶基因有beclin 1和sestrin 2。构建含beclin 1和sestrin 2作用位点的GFP载体共5个,质粒转染Hela细胞后在荧光显微镜下可见绿色荧光。结论成功构建hsa-miR-34a下游自噬相关基因靶位点序列GFP报告基因载体,为进一步研究hsa-miR-34a对自噬的调控作用奠定基础。     

人肝细胞自噬性凋亡的形态学观察

为了进一步完善形态学的分型标准以及研究调控自噬对细胞凋亡的影响 ,采用去血清诱导自噬和3ＭＡ抑制自噬 ,同时施与凋亡诱导剂 ,观察细胞超微结构的改变。正常人肝ＬＯ2细胞株去血清培养12ｈ诱导自噬之后 ,ｃｉｓｐｌａｔｉｎ(3μｌ ｍｌ)加入 16 40培养基诱导ＬＯ2细胞凋亡 16ｈ和 3-ＭＡ自噬抑制剂(6 0 0 μｇ ｍｌ) ,30ｍｉｎ后加入ｃｉｓｐｌａｔｉｎ(3μｌ ｍｌ)混合培养ＬＯ2细胞 16ｈ。ＪＥＭ 12 0 0ＥＸ透射电镜观察、摄片。正常ＬＯ2细胞胞质丰富 ,核仁明显 ,自噬现象少见。单施加凋亡诱导剂的细胞 ,可见凋亡细胞的形态多样 ,细胞…     

腹股沟肉芽肿的超微结构观察

目的:探讨腹股沟肉芽肿的组织结构和超微结构病变特征.方法:对2例腹股沟肉芽肿患者的病变组织进行光镜和透射电镜观察.结果:光镜下,肉芽肿组织内有大量巨噬细胞,其胞质内可查见革兰氏阴性的肉芽肿荚膜杆菌;透射电镜下巨噬细胞胞质内可见较大的初级溶酶体和大量形态各异、大小不等的次级溶酶体和板层样膜状结构的吞噬体(即杜诺凡小体),偶见吞噬体内有菌体剖面或其残片.结论:杜诺凡小体是巨噬细胞吞噬肉芽肿荚膜杆菌后在胞质溶酶体内形成的形态各异、大小不等的吞噬体,其超微结构形态学特征,具有临床诊断的意义.     

基底细胞上皮瘤细胞内黑素小体复合体形成的超微结构观察

应用透射电镜观察了21例基底细胞上皮瘤,结果显示基底细胞上皮瘤中有许多不同阶段的凋亡肿瘤细胞,部分被相邻肿瘤细胞吞噬,被吞噬消化的凋亡细胞中黑素小体的结构基本保持完整,而其他细胞器则失去正常结构。相邻肿瘤细胞对凋亡细胞的循环吞噬最终在肿瘤细胞内形成黑素小体复合体,这可能是黑素细胞向肿瘤细胞输入黑素小体的重要途径之一。     

彩虹明樱蛤精子发生的超微结构

应用透射电镜观察彩虹明樱蛤的精子发生过程,描述了从精原细胞发育成为成熟精子过程中的超微结构变化。主要表现在染色质以颗粒状形式浓缩和分散;高尔基体分泌的前顶体颗粒逐渐融合形成前顶体囊、前顶体最终发育成为顶体;线粒体逐渐融合形成精子中段;细胞核的拉伸和旋转以及中心体和鞭毛的形成等。在初级精母细胞阶段,同源染色体经历了联会复合体形成和解体的变化。前顶体颗粒和中心粒结构在精原细胞期就已经存在。根据染色质和顶体发育的变化特点将精细胞分为6个时期,并描述了各期精细胞的超微结构特点。彩虹明樱蛤成熟精子属于原生型,由头部、中段和尾部组成。     

Synergy of lysozyme and lanoconazole on the morphology of Candida albicans

Synergism of lysozyme with the imidazole antifungal lanoconazole (LCZ) has been demonstrated in vitro against Candida albicans. To determine the mechanism of the synergistic action, the effects of subinhibitory concentrations of lysozyme (50 microg ml(-1)) and LCZ (1.25 microg ml(-1)), used alone and in combination, on the morphology and ultrastructure of C. albicans cells were studied by fluorescence microscopy using Fungiflora Y, as well as by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In the presence of lysozyme, the separation of apparently mature cells from each other was inhibited, and cells in which cell wall-like material had markedly accumulated in the periplasmic space were frequently observed. Such intracellular ultrastructural changes were not seen in LCZ-treated cells, but normal septal wall biogenesis was inhibited with the resultant formation of long chains of interconnecting cells. When the cultures were treated with the combination of lysozyme and LCZ, intracellular accumulation of cell wall-like materials increased in extent, and the peripheral wall structure was severely damaged. These morphological and ultrastructural findings lead us to the following conclusions: (1) lysozyme and LCZ inhibit the process of cell division in different manners; (2) lysozyme appears to affect cell wall formation more directly and drastically by interrupting the normal assembly and integration of cell wall components; and (3) these effects of lysozyme are noticeably enhanced when combined with LCZ.     

蒙药鹅绒藤扫描电镜的观察研究

目的：为蒙药鹅绒藤药材鉴定研究提供地上部分表面的微形态依据。方法：采用扫描电子显微镜技术进行观察研究。结果：鹅绒藤叶片上下表皮、内外果皮、种子背腹面和花粉块的微形态各异。结论：为鹅绒藤药材微形态鉴定和生物学研究提供参考依据。     

Ultrastructural anomalies of the desmosomal contacts in monolayer transformed cultures

The ultrastructure of cell-cell desmosomal contacts and their cytoskeletal components in the cellular monolayer of transformed epithelial cultures is studied. It is suggested that asymmetry of the desmosomal contact formation, the formation of hemidesmosome-like structures on the lateral surface of one of the contacted cells and defects of cytoskeletal desmosomal components are related to transformational changes in the functionally different contacted cells.     

Three-dimensional structure of intracellular membranous organelles in embryonic chick skeletal muscle cells in vitro revealed with scanning electron microscope

Using the freeze-polishing and osmium-maceration procedures, the ultrastructure of intracellular membranous organelles in embryonic chick skeletal muscle cells in vitro was investigated three-dimensionally with the scanning electron microscope (SEM). T-system tubules followed a tortuous course and possessed many warts. Some of these tubules could be traced to the sarcolemma. The sarcoplasmic reticulum (SR) had elaborate structures of a hexagonal pattern. By comparing the SEM images with those of ultrathin sections of myotubes impregnated with ferritin particles, the distinction between the T-system and SR was confirmed. Late in the culture, these two intracellular membranous systems were arranged periodically at right angles to the longitudinal axis of the myotubes. Round-shaped mitochondria were often arranged in chains connected with slender rods. Long rod-shaped mitochondria exhibited occasional branchings.     

胆结石的微观结构和组成元素研究

用扫描电镜研究了胆色素型和混合型胆结石经氯仿、乙醇、乙醚、盐酸等溶剂溶解剩余物的微观结构，结果显示色素型结石难溶物为膜孔相连结构，两层膜之间填充颗粒状盐；混合型结石难溶物的微观结构为絮状物连接的网孔结构，盐类嵌镶在絮状物中。用X射线能谱仪和等离子体发射光谱仪分析了胆结石的元素，讨论了金属元素在结石中所起的作用。     

快速静脉注射对小鼠肝细胞超微结构的影响

目的：观察快速静脉注射对小鼠肝细胞超微结构的影响。方法：通过小鼠尾静脉以不同速度快速注射大体积的生理盐水，在注射后不同时间经眼眶取血，检测血清中丙氨酸氨基转移酶（ALT）和天门冬氨酸氨基转移酶（AST）水平；取小鼠肝脏在透射电镜下观察肝细胞超微结构的改变。结果：电镜下8s注射组在注射后10h。肝细胞内可见线粒体轻度肿胀，3天后基本恢复到正常超微结构。3s注射组肝细胞胞浆内糖原颗粒减少出现空化区，部分肝细胞线粒体内可见灶性絮状致密区，7天后基本恢复到正常超微结构。尾静脉注射生理盐水后10h，8s注射组和3s注射组小鼠血清中ALT、AST水平均最高，24h后均开始快速下降，7天后ALT、AST恢复至正常水平。结论：快速静脉注射可以引起肝细胞超微结构损伤，损伤程度与注射速度有关。     

骨巨细胞瘤组织及原代培养细胞超微形态学观察

目的：通过骨巨细胞瘤组织及原代培养细胞与其超微形态学观察,了解该肿瘤的体外生长行为、特点及细胞细胞类型进一步探讨其组织来源。方法：对收集到的８例切除或刮除骨巨细胞瘤标本进行了原代培养。所有标本术前均未接受放疗或化疗;术后经病理证实,实体瘤及培养细胞均按常规方法制备标本,透射电镜观察。结果：本组原代培养成功６例,培养细胞从形态上主要可见到多核巨细胞、梭形的单个核细胞、单相核吞噬细胞样细胞。电镜观察发现无论是组织样本还是细胞样本,单个核细胞与多核巨细胞的形态相似,二者的超微结构形态未见明显差异;培养细胞与组织样本中所见的瘤细胞形态未见差别,而瘤细胞形态与正常间质纤维母细胞完全不同,说明培养细胞主要为肿瘤性细胞。结论：原代培养存活期较长的细胞主要为梭形细胞,这些细胞与实体瘤组织中的梭形细胞形态一致,支持梭形细胞为骨巨     

Cytochemistry of gastric parietal cells with high-pressure freezing followed by freeze-substitution

Gastric parietal cells were examined for changes in their ultrastructure and distribution of the proton pump during feeding and fasting states in rats. The fundic glands from rats fed ad libitum or fasted with free access to water were cryofixed using high-pressure freezing followed by freeze-substitution in acetone containing osmium or acrolein and then embedded in Epon 812 or Lowicryl K4M resin, respectively. Excellent ultrastructural preservation was achieved. During the feeding state, intracellular canaliculi and numerous microvilli were well developed, while tubulovesicles were poorly developed. In contrast, during the fasting state, the microvilli in the narrowed space of the intracellular canaliculi were tightly packed and the tubulovesicles were enlarged. Ultrathin sections were immunostained with antibodies against the alpha- and beta-subunits of the proton pump, H+ x K(+)-ATPase, using the immunogold method. The labelling was strong and clearly localized in comparison with that obtained using the conventional chemical-fixation method. Each subunit was localized on the membrane of the microvilli, intracellular canaliculi and tubulovesicles. The distribution of subunit proteins varied between the two states. During ad libitum feeding, the immunolabelling was localized strongly on the membranes of the microvilli and intracellular canaliculi. In contrast, the labelling was strong on the tubulovesicle membrane in the fasting state. The results obtained with each anti-subunit antibody by H+ x K(+)-ATPase immunostaining revealed differences in distribution and labelling density between the feeding and fasting states.     

A Zn‐Doped CuO Nanocomposite Shows Enhanced Antibiofilm and Antibacterial Activities Against Streptococcus Mutans Compared to Nanosized CuO

Zinc‐doped copper oxide and copper oxide nanoparticles (NPs) are synthesized and deposited on artificial teeth by sonic irradiation, and the ability of these coatings to restrict biofilm formation by Streptococcus mutans is examined. The CuO and Zn:CuO NP‐coated teeth show significant reductions in biofilm formation of 70% and 88%, respectively, compared to uncoated teeth. The mechanism of the Zn:CuO nanoparticles is investigated, revealing that the nanoparticles attach to and penetrate the bacteria and generate intracellular reactive oxygen species (ROS) that enhance lipid peroxidation and cause cell death. Conversely, the CuO or ZnO NPs do not show this behavior and could not generate intracellular ROS. These results highlight the superior efficacy of Zn:CuO nanocomposites over CuO and ZnO NPs and the role of ROS in their antimicrobial effect.