变异链球菌生物膜结构观察

目的　建立变异链球菌生物膜模型 ,用激光共聚焦扫描显微镜 (CLSM)观察变异链球菌生物膜结构。方法　在盖玻片上分别形成 6、12、18、2 4、4 8、72h变异链球菌生物膜 ,将得到的各时段生物膜荧光染色后 ,用CLSM观察生物膜的断层扫描图像、生物膜厚度、每层红光绿光的面积 ,计算生物膜中细菌密度和活菌百分比 ,用软件处理扫描数据 ,得到生物膜的三维重建图像。结果　变异链球菌生物膜具有空间立体结构 ,形态多样 ,其中细菌密集 ,由死细菌和活细菌组成 ,还有丰富的基质和管道系统。 2 4h生物膜平均厚度最大 ,生物膜内层、中间层的细菌密度相对较大 ,而外层较低 ,72h内各时间段生物膜中活菌百分比由内往外逐渐增加。结论　变异链球菌生物膜有一定的厚度 ,具有三维立体空间结构 ,结构形态具有多样、不均质、开放的特点。     

Development of myocardial fiber organization in the rat heart

Confocal laser-scanning microscopy of phalloidine-stained actin fibers is a relatively new tool for studying the development of myocardial fiber organization. It seems to show orientation of myocytes in rather early embryonic stages. To further evaluate the differentiation of the myocardium, this technique was compared with transmission electron microscopy in rat embryos aged between 11 and 18 days. Although the confocal images of actin filament patterns pointed to early myocyte orientation, the electron micrographs revealed that even at 17 days the ventricular myocardium was far from mature. Myofibrils never completely filled the myocytes, and lack of organization was the rule rather than the exception. The organized structure as revealed by confocal microscopy was based on cell-to-cell continuity, whereas electron microscopy showed crossing and disarray within individual myocytes. Exceptions were in the ventricular trabeculations, which showed precocious myofiber differentiation. The trabeculations probably support ventricular systole in those stages in which the free walls do not yet provide efficient contractions. The other exception was the wall of the outflow tract, which showed well-oriented myofibrils from early stages onwards. Apparently, the outflow tract has a different function in these stages. The differences found between confocal microscopy and electron microscopy suggest that some caution is indicated in the interpretation of fluorescent images of relatively low magnification.     

Histamine-immunoreactive neurons in the brain of the teleost Gasterosteus aculeatus L. Correlation with hypothalamic tyrosine hydroxylase- and serotonin-immunoreactive neurons

The distribution of putative histaminergic neurons in the brain of a teleost, the three-spined stickleback, was investigated by means of immunocytochemistry using specific antibodies against histamine (HA), and conventional microscopy as well as confocal laser scanning microscopy. Histamine-immunoreactive (HAir) neurons form discrete populations ventral to the nucleus of the posterior recess (NRP) and in the nucleus saccus vasculosus (NSV), which belong to the periventricular hypothalamic nuclei. The neuronal somata are subependymally located, and do not possess apical neurites contacting the cerebrospinal fluid. They give rise to both long-range and local axonal projections. The local projections give rise to a field of dense punctate immunoreaction dorsal to the NRP and lateral to the NSV. Long-range projections are comprised of ascending projections to the thalamus, habenula, preoptic area and dorsal telencephalon; and descending projections via the posterior tuberal nucleus, ventrally to the nucleus interpeduncularis, and dorsally into the central gray. HAir neurons occur together with serotoninergic cerebrospinal fluid-contacting (CSFc) neurons in the NRP, and with tyrosine hydroxylase-immunoreactive (THir) neurons in the NSV. Although HAir elements occur together with THir ones in many brain areas, direct contacts between the two neurotransmitter systems are rare. The putative histaminergic neurons in the brain of the three-spined stickleback constitute a very discrete neuronal system, with a major projection area in the dorsal telencephalon in a region which is considered homologous with the dorsal pallium of land vertebrates.     

探索双重荧光染色并利用两种不同显微镜在Hep-2细胞上观察抗核抗体荧光模式的临床价值

目的 探索荧光显微镜,激光扫描共聚焦显微镜以及双重荧光染色法在Hep-2细胞上观察抗核抗体荧光模式的临床价值.方法 选取临床上抗核抗体呈阳性的血清样本结合到抗原片上,先后用异硫氰酸荧光素(FITC)和4 ',6-二脒基-2-苯基吲哚(DAPI)两种荧光染料孵育,之后在两种不同的显微镜下观察.结果 FITC荧光出现的部位是抗原抗体结合处,DAPI荧光出现的部位为细胞核,当通过DAPI荧光对细胞核定位后,可使得FITC的荧光模式更易于观察.本实验通过利用两种荧光标记的方法在两种显微镜上分别观察了斑点型、均质型、着丝点型、核点型、核膜型、胞浆颗粒型——抗高尔基体抗体.结论 综合各种因素,利用双荧光染色法染色,并在激光扫描共聚焦显微镜下观察,对于在Hep-2细胞上观察抗核抗体荧光模式最具临床价值.     

mSemcap2对HeLa细胞骨架及福氏志贺菌入侵HeLa细胞的影响

目的 研究mSemcap2对HeLa细胞骨架的影响及其对福氏志贺菌（F2a)入侵HeLa细胞的影响，以了解mSemcap2的功能。方法 利用免疫荧光细胞化学染色结合激光共聚焦显微镜观察转染重组质粒的HeLa细胞和野生HeLa细胞系微丝及微管的含量及分布；利用普通光学显秽间及激光共聚焦显微镜观察F2a对2种细胞入侵的差别。结果 转染mSemcap2后的HeLa细胞，其F-actin呈解聚状态，荧光强度减弱，actin在细胞核周集中分布的现象消失；转染mSemcap2后的HeLa细胞，其F-actin呈解聚状态，荧光强度减弱，actin在细胞核周集中分布的现象消失；转染mSemcap2的HeLa，其F2a入侵率明显高于野生HeLa细胞。结论 mSemcap2可以通过细胞骨架的重排来促进细菌对上皮细胞的入侵。     

5-ALA诱导白念珠菌生成PpIX与ALA浓度相关性研究

目的 对不同浓度光敏剂5-氨基酮戊酸(5-ALA)在白念珠菌体内生成原卟啉IX (PpIX)进行荧光强度测定,探求PpIX荧光强度与ALA浓度的关系.为光动力治疗白念珠菌选择最佳的光敏剂浓度提供理论依据.方法 实验分为实验组和对照组,将各组白念珠菌悬液与不同浓度5-ALA混合后避光60min孵育.利用共聚焦激光扫描显微镜测量PpIX荧光强度.结果 实验组白念珠菌与不同浓度ALA避光孵育后均有荧光物质PpIX产生,200mg/mL以上ALA浓度组产生的PpIX强度无统计学意义.300mg/mL以下ALA浓度与PpIX生成强度有相关性.对照组没有检测到PpIX.结论 ALA浓度与生成的PpIX荧光强度密切相关.这为临床ALA-PDT治疗白念珠菌疾病提供了实验依据.     

Bitter taste and blood glucose are not involved in the suppressive effect of dietary histidine on food intake

Natriuretic peptides (NPs) may act as neuromodulators through activation of three specific receptor subtypes (NPRs). In the present study we examined the expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) on different subtypes of retinal amacrine cells (ACs) in rat by immunofluorescence double labeling. All three NPs were moderately expressed in dopaminergic and cholinergic ACs, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), respectively. The immunostaining appeared on the membrane, cytoplasm and somatodendritic compartments of these ACs. In AII glycinergic ACs, labeled by parvalbumin (PV), however, only faint punctate staining, if any, was seen. These results suggest that NPs could be produced in ACs and play a neuromodulatory role in the inner retina. Together with a previous immunocytochemical study, showing that NPR-B is present in cultured rat GABAergic ACs, our results further suggest that NPs produced in ACs may also modulate their own activity.     

滑膜细胞中波形蛋白的形态学研究

用免疫组织化学、激光共聚焦扫描和粘附式细胞仪上进行三维图像重建，观察了波形蛋白在体外培养人腱鞘滑膜细胞内的分布。结果显示：波形蛋白呈海绵状立体结构，分布于整个细胞质空间；认为波形蛋白的分布与培养细胞在不同生长时期的形态变化密切相关。     

Image cross-correlation spectroscopy: A new experimental biophysical approach to measurement of slow diffusion of fluorescent molecules

The technique of image cross-correlation spectroscopy has been applied to measurements of diffusion of fluorescent beads in polymer solution. The technique is based on measurement and analysis of temporal fluctuations of the intensity observed in fluorescence confocal microscope images. As with other fluctuation techniques, fluorescence fluctuations arise from stochastic concentration fluctuations about the equilibrium concentration. The dynamics of the fluctuations depend on the number of fluorescent molecules in the observation volume. This work presents the theory and illustrates how the technique can be applied to measurement of diffusion of fluorescent beads in solutions of various viscosities. Further we extend the concept of cross-correlation to studies on the cell surface to detect the relative distribution of molecules (receptors) on the cell surface, in which intensity fluctuations arising from samples containing two distinct types of labelled molecules are cross-correlated using two detectors with different wavelength sensitivities.     

三种含硒化合物对K562细胞的抑制作用

目的 研究比较3种新的含硒化合物在体内、外的抗癌作用和作用机制. 方法 通过四甲基偶氮唑盐(MTT)比色法和移植瘤生长抑制实验及形态学观察、流式细胞术以及激光扫描共焦显微镜,检测分析含硒化合物对K562细胞的抑制作用. 结果 3种含硒化合物能明显抑制K562细胞增殖(P<0.05)和S180、H22移植瘤(n=10)的生长(P<0.01);使K562细胞出现体积缩小,细胞膜完整,染色质高度凝集,边集,核浓染、碎裂,伴有出泡现象和凋亡小体出现等典型的凋亡特征;对K562细胞的周期分布有明显影响,且在大剂量时出现了明显的亚二倍体峰;能够明显增加K562细胞内Ca2+、Mg2+和细胞内活性氧(ROS)的荧光强度(P<0.01),但pH值和线粒体膜电位显著降低(P<0.01). 结论 3种含硒化合物均有体内、体外抗肿瘤作用,其作用机制可能与Ca2+、Mg2+、(活性氧ROS)、pH值和线粒体膜电位(MMP)诱导的细胞凋亡有关.