心钠素对培养人心包间皮细胞内钙离子浓度的影响

目的　探讨心纳素与人类心包间皮细胞之间的关系。　方法　分离人心包间皮细胞进行体外培养 ,用Fluo 3作为钙的指示剂 ,利用激光共聚焦显微镜测定 10 - 9mol L ,10 - 1 0 mol L ,10 - 1 1 mol L心钠素 (ANP)分别用作用于人心包间皮细胞后细胞内Ca2 (Ca2 )浓度变化。　结果　人心包间皮细胞存在细胞内Ca2 浓度的改变 ,随着ANP浓度的增加 ,细胞内Ca2 浓度出现非常明显的变化 (P <0 0 0 0 1) ;细胞内Ca2 浓度随时间变化也出现明显的变化 (P <0 0 0 0 1) ;不同时间测定的钙离子浓度与ANP浓度之间存在着交互作用 (P <0 0 0 0 1)。　结论　人心包间皮细胞存在细胞内Ca2 的改变 ;不同浓度的ANP作用于间皮细胞后引起细胞内Ca2 浓度出现明显不同的改变     

肝癌细胞纤维肌动蛋白的共聚焦激光扫描显微镜光学切片观察

目的：建立培养细胞共聚焦激光扫描显微镜光学切片的方法。探讨肝癌细胞纤维肌动蛋白（F-actin)的空间结构。方法：采用共聚焦激光扫描显微镜光学切片技术结合异硫酸氢荧光素-鬼笔环肽（FTTC-phalloidin)标记纤维肌动蛋白和碘化丙啶（PI）标记细胞核的荧光探针双重标记技术对肝癌细胞纤维肌动蛋白进行形态学观察和图像分析。结果：肝癌细胞内纤维肌动蛋白微丝形成束状纤维。粗壮而密集,平行排列或纵横交错成网状贯穿整个细胞和细胞突起。结论：（1）共聚焦激光扫描显微镜的光学切片技术结合FTTC-phalloidin和PI荧光探针双重标记技术是观察和分析细胞骨架系统三维立体结构的良好方法;（2）肝癌细胞内纤维肌动蛋白微丝粗壮、形成束状或网状结构。     

Measuring enamel erosion: A comparative study of contact profilometry,non-contact profilometry and confocal laser scanning microscopy

Objectives

To compare three instruments for their ability to quantify enamel loss after acid erosion.

Methods

6 randomized parallel groups of bovine enamel samples were subjected to citric acid (higher acidity) or orange juice (lower acidity) erosion and remineralisation in a cycling model. Two protected shoulders were created on each of the samples using tape, to serve as reference for analysis. The time of exposure to each acid was varied, along with presence or absence of agitation. After treatment, samples were measured on 3 instruments capable of measuring step height: a contact profilometer (CP); a non-contact profilometer (NCP); and a confocal laser scanning microscope (CLSM) by three different examiners. Additionally, 3D (volume) step height was also measured using the CLSM.

Results

Increasing acid concentration and exposure time resulted in greater erosion, as did agitation of samples while in acid solution. All instruments/methods identified the same statistically significant (p < 0.05) pair-wise differences between the treatments groups. Further, all four methods exhibited strong agreement (Intra-class correlation ≥ 0.96) in erosion level and were highly correlated, with correlations of 0.99 or higher in all cases.

Significance

All instruments/methods used in this study produced very similar conclusions with regard to ranking of enamel loss, with data showing very high agreement between instruments. All instruments were found to be equally suited to the measurement of enamel erosion.     

Colonisation of gingival epithelia by subgingival biofilms in vitro: Role of “red complex” bacteria

Objectives

Biofilm formation on tooth surface results in colonisation and invasion of the juxtaposed gingival tissue, eliciting strong inflammatory responses that lead to periodontal disease. This in vitro study investigated the colonisation of human gingival multi-layered epithelium by multi-species subgingival biofilms, and evaluated the relative effects of the “red complex” species (Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola).

Methods

The grown biofilm consisted of Fusobacterium nucleatum, Campylobacter rectus, Veillonella dispar, P. gingivalis, Prevotella intermedia, T. forsythia, T. denticola, Actinomyces oris, Streptococcus anginosus and Streptococcus oralis, or its variant lacking the “red complex”. After 48 h in co-culture with the gingival epithelia, the bacterial species in the biofilm were quantified, whereas their localisation on the cell surface was investigated by combining confocal-laser scanning microscopy (CLSM) and fluorescence in situ hybridisation (FISH), as well as by scanning electron microscopy (SEM).

Results

Exclusion of the “red complex” quantitatively affected S. oralis, but not other species. The “red-complex” species were all able to colonise the gingival epithelial cells. A co-localisation trend was observed between P. gingivalis and T. denticola, as determined by FISH. However, in the absence of all three “red complex” bacteria from the biofilm, an immense colonisation of streptococci (potentially S. oralis) was observed on the gingival epithelia, as confirmed by both CLSM and SEM.

Conclusions

While the “red complex” species synergise in colonizing gingival epithelia, their absence from the biofilm enhances streptococcal colonisation. This antagonism with streptococci reveals that the “red complex” may regulate biofilm virulence, with potential implications in periodontal pathogenesis.     

粪肠球菌ace阳性和阴性菌株体外生物被膜形成能力比较

目的 比较粪肠球菌ace基因阳性、阴性菌株的生物被膜形成能力,探讨粪肠球菌ace基因是否有利于细菌生物被膜的形成。方法 微量滴定板法检测46株临床分离粪肠球菌的生物膜形成能力,PCR方法检测其ace基因,比较生物膜阳性组、阴性组粪肠球菌ace基因检出率;将粪肠球菌ATCC29212(ace⁺)、野生株U8-ace^-、空质粒对照株EU8-ace^-、转化株ZU8-ace⁺接种到96孔板,培养3 h、6 h、12 h、24 h、36 h、48 h,结晶紫染色,测定OD₅₇₀,微量滴定板法比较ATCC29212(ace⁺)、U8-ace^-、EU8-ace^-以及ZU8-ace⁺的生物被膜形成能力;将ATCC29212(ace⁺)、U8-ace^-、EU8-ace^-、ZU8-ace⁺在含盖玻片的细胞培养皿内培养6 h~72 h,共聚焦激光扫描显微镜(CLSM)观察比较ace⁺、ace^-粪肠球菌形成的生物膜平均厚度和密度。结果 生物膜阳性组粪肠球菌ace基因检出率为76.67%,高于生物膜阴性组31.25%的检出率,差别有统计学意义 (χ²= 9.04,P> 0.05);微量滴定板法显示不同时间点ATCC29212(ace⁺)以及转化株ZU8-ace⁺的OD₅₇₀值都大于U8-ace^-的OD₅₇₀值,P< 0.01,而EU8-ace^-与U8-ace^-比较,OD₅₇₀值无统计学差别,P> 0.05;CLSM观测的ace⁺ 粪肠球菌在不同阶段所形成的生物膜的平均厚度、密度均高于ace^-粪肠球菌,P< 0.01,空质粒对照株与野生株比较,生物膜的厚度、密度无统计学差别,P> 0.05。结论 粪肠球菌胶原黏附素ace基因或有利于肠球菌生物被膜的形成。     

Evidence from Confocal Fluorescence Microscopy for a Dense, Reciprocal Innervation Between AVP-,somatostatin-, VIP/PHI-, GRP- and VIP/PHI/GRP-immunoreactive Neurons in the Rat Suprachiasmatic Nucleus

The rat suprachiasmatic nucleus (SCN) consists of several classes of neurons which can be identified by their transmitter content. Knowledge of putative interaction between these different cell types is essential in order to understand the possibilities of information processing within the SCN. The aim of the present study was therefore to obtain more information about the mutual innervation between the main cell classes in the rat SCN, viz. those containing the neuropeptides arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), gastrin-releasing peptide (GRP) and somatostatin respectively. For this purpose, vibratome sections were double-immunolabelled for seven different peptide combinations and subsequently analysed by high-resolution confocal laser scanning fluorescence microscopy. Attention was focused on axosomatic appositions, the occurrence and frequency of which were quantitatively estimated. Our analysis of double-immunolabelled sections demonstrated that some of the VIP- and some of the GRP-immunoreactive nerve cells and endings showed colocalization. Assuming, on the basis of literature data, that VIP and PHI are always colocalized at the cellular level, the five main cell classes in the SCN appeared to be interconnected, at least axosomatically, in the following reciprocal way: AVP ? VIP/PHI, AVP ? GRP, AVP ? somatostatin, somatostatin ? VIP/PHI, somatostatin ? GRP, VIP/PHI ? GRP, VIP/PHI/GRP ? GRP, VIP/PHI/GRP ? VIP/ PHI. In addition to this heterologous axosomatic innervation, these cell groups also showed substantial homologous innervation. Supported by electron microscope data from the literature showing the existence of axodendritic synapses for some of these peptide combinations, our findings strongly suggest that the rat SCN comprises a complex synaptic network with strong interactive capabilities, which is probably a requisite for its biological clock function.     

Confocal laser scanning microscopy used to monitor intracellular Ca changes in hippocampal CA 1 neurons during energy deprivation

An increase in intracellular calcium during cerebral ischemia has been proposed as a common final pathway underlying the events leading to neuronal death. Intracellular calcium has been measured with ion selective electrodes during energy deprivation (ED) in hippocampal slices and with fluorescent techniques in neuronal cultures. In the present study, we describe a novel method to visualize and quantify changes in intracellular calcium in brain slices using Confocal Laser Scanning Microscopy (CLSM). CA 1 pyramidal neurons in hippocampal slices were filled by intracellular injection with a 1:2 mixture of the fluorescent dyes Fluo 3 and Fura Red. The neurons were then visualized using CLSM, and the ratio of the fluorescence from each probe used to quantify intracellular calcium concentrations before and during ED. The free intracellular calcium concentration was 60 nM prior to ED and increased to 24 μM during ED. These results demonstrates that CLSM and fluorescent probes can be used in functional neuronal networks in addition to cell cultures as previously described.     

Effect of two antimicrobial agents on early in situ biofilm formation

OBJECTIVES: The aim of this observer-blind, controlled, three-cell cross-over study was to evaluate the influence of an amine fluoride/stannous fluoride (Meridol, 250 ppm; ASF) and a chlorhexidine mouthrinse (CHX; Chlorhexamed forte, 0.2%) compared with water on in situ biofilm growth. MATERIAL AND METHODS: After a professional toothcleaning seven volunteers had to wear a special acrylic appliance, in which six specimens each were inserted to allow the build-up of intra-oral biofilms. The volunteers had to rinse twice daily for 1 min. with 10 ml of the allocated mouthrinse. After 48 h of wearing, the specimens with the adhering biofilms were removed from the splints and stained with two fluorescent dyes, which selectively stain vital bacteria green and dead bacteria red. Under the confocal laser scanning microscope biofilm thickness (BT) was evaluated. To examine bacterial vitality (BV%) the biofilms were scanned (1 microm sections) and digital images were made. An image analysis program was used to calculate the mean BV as well as the BV of the single sections. After a wash-out period of 14 days a new test cycle was started. RESULTS: The use of CHX and ASF resulted in a BT of 8.4+/-4.4 mum and 15.7+/-9.9 compared with 76.7+/-29.4 mum using water. The mean vitality (in %) was reduced from 66.1+/-20.4 to 23.3+/-11.6 and 23.9+/-12.4 using CHX and ASF, respectively. Both active solutions reduced BT and BV significantly compared with water (p<0.001). Differences between the two active solutions were not significant (p>0.05). CONCLUSION: Both mouthrinses showed antibacterial and plaque-reducing properties against the in situ biofilm. The study design enables the examination of an undisturbed oral biofilm and for the first time shows the influence of antibacterial components applied under clinical conditions regarding biofilm formation.     

Novel non-uniform distribution of serotonin transporter in the mouse hippocampus and neocortex revealed by N- and C-terminal domain-specific immunohistochemistry

Serotonergic fibers have a general feature of extending diffusely throughout the brain and appear to innervate broad areas rather uniformly. The present study revealed marked regional difference in their immunoreactivities against serotonin transporter by using two antibodies that recognize either N- or C-terminal domain of the transporter. C-terminal-specific labeling was ubiquitous, whereas N-terminal-specific labeling was confined to hippocampal CA1 region, somatosensory cortex, and other areas, suggesting novel non-uniformity in the serotonergic system.