肝细胞肝癌中卵圆细胞的组织学与超微结构研究

目的：探讨人肝癌肝组织存在卵圆细胞的可能性。方法：对２０例人肝细胞肝癌的手术标本行常规组织学和超微结构观察，并用胆管上皮分化标记ＣＫ７和肝细胞分化标记白蛋白对以上组织作免疫组化染色，同时对其中５例作免疫电镜标记。结果：光镜下，１４／２０例癌肿边缘常可见到增生的小胆管样结构。电镜下，１４／２０例可找到三型卵圆细胞。其中Ⅰ型细胞体积较小、核大、胞质少，此为较为原始的卵圆细胞。Ⅱ型细胞体积稍大，胞质稍多     

Overcoming the Challenges of Detecting GPCR Oligomerization in the Brain

G protein-coupled receptors (GPCRs) constitute the largest group of membrane receptor proteins controlling brain activity. Accordingly, GPCRs are the main target of commercial drugs for most neurological and neuropsychiatric disorders. One of the mechanisms by which GPCRs regulate neuronal function is by homo- and heteromerization, with the establishment of direct protein-protein interactions between the same and different GPCRs. The occurrence of GPCR homo- and heteromers in artificial systems is generally well accepted, but more specific methods are necessary to address GPCR oligomerization in the brain. Here, we revise some of the techniques that have mostly contributed to reveal GPCR oligomers in native tissue, which include immunogold electron microscopy, proximity ligation assay (PLA), resonance energy transfer (RET) between fluorescent ligands and the Amplified Luminescent Proximity Homogeneous Assay (ALPHA). Of note, we use the archetypical GPCR oligomer, the adenosine A_2A receptor (A_2AR)-dopamine D₂ receptor (D₂R) heteromer as an example to illustrate the implementation of these techniques, which can allow visualizing GPCR oligomers in the human brain under normal and pathological conditions. Indeed, GPCR oligomerization may be involved in the pathophysiology of neurological and neuropsychiatric disorders.     

肾轻链沉积病和轻链型淀粉样变的电镜及免疫电镜研究

目的：探讨电镜及免疫电镜方法在肾轻链沉积病(light chain deposition disease，LCDD)和轻链型淀粉样变(amyloid，AL)诊断中的作用。方法：对我院1994年1月至2002年2月间的肾活检病例进行电镜观察，对疑为LCDD及早期AL的病例进行轻链(κ、λ)的免疫电镜标记。结果：6例符合LCDD的诊断，36例符合AL的诊断。LCDD电镜下可见肾小管基底膜外侧、肾小球基底膜内侧、鲍曼囊外侧及小血管壁的点状、颗粒状电子致密物质沉积，其中2例早期病例表现颗粒状物质节段性分布；免疫电镜标记，颗粒状物质被单种轻链标记，其中K轻链4例，λ轻链2例。AL电镜下可见肾小球系膜区、毛细血管壁及小动脉壁的纤维物质分布，其中15例早期病例表现系膜区或／及毛细血管壁的纤维结构呈节段性分布；15例早期AL的免疫电镜标记结果，纤维结构被单种轻链标记，其中κ轻链4例，λ轻链11例。结论：肾脏LCDD及早期AL的诊断需依赖于电镜检查，免疫电镜检查对早期LCDD及早期AL的确诊具有不可替代的作用。     

Cellular and subcellular localization of alpha-1 adrenoceptors in the rat visual cortex

Noradrenaline is thought to play modulatory roles in a number of physiological, behavioral, and cellular processes. Although many of these modulatory effects are mediated through alpha-1 adrenoceptors, basic knowledge of the cellular and subcellular distributions of these receptors is limited. We investigated the laminar distribution pattern of alpha-1 adrenoceptors in rat visual cortex, using immunohistochemistry at both light and electron microscopic levels. Affinity-purified anti-alpha-1 antibody was confirmed to react only with a single band of about 70-80 kDa in total proteins prepared from rat visual cortex. Alpha-1 adrenoceptors were widely distributed though all cortical layers, but relatively high in density in layers I, II/III, and V. Immunoreactivity was observed in both neuronal perikarya and processes including apical dendrites. In double-labeling experiments with anti-microtubule-associated protein 2, anti-neurofilament, anti-glial fibrillary acidic protein, anti-glutamic acid decarboxylase 65/67, anti-2-3-cyclic nucleotide 3-phosphodiesterase, and anti-tyrosine hydroxylase antibodies, alpha-1 adrenoceptors were found mainly in dendrites and somata of microtubule-associated protein 2-immunopositive neurons. About 20% of alpha-1 adrenoceptors were in GABAergic neurons. A small number of alpha-1 adrenoceptors were also distributed in axons of excitatory neurons, astrocytes, oligodendrocytes and noradrenergic fibers. Using an immunoelectron microscopic technique, numerous regions of alpha-1 adrenoceptor immunoreactivity were found in cell somata, on membranes of dendrites, and in postsynaptic regions. Moreover, a small number of immunoreaction products were also detected in axons and presynaptic sites. These findings provide the first quantitative evidence regarding the cellular and subcellular localization of alpha-1 adrenoceptor immunoreactivity in visual cortex. Moreover, the ultrastructural distribution of alpha-1 adrenoceptor immunoreactivity suggests that alpha-1 adrenoceptors are transported mainly into dendrites and that they exert effects at postsynaptic sites of neurons.     

Localization and physiological regulation of the exocytosis protein SNAP-25 in the brain and pituitary gland of Xenopus laevis

In mammals, the synaptosomal-associated protein of 25 kDa, SNAP-25, is generally thought to play a role in synaptic exocytosis of neuronal messengers. Using a polyclonal antiserum against rat SNAP-25, we have shown the presence of a SNAP-25-like protein in the brain of the South-African clawed toad Xenopus laevis by Western blotting and immunocytochemistry. Xenopus SNAP-25 is ubiquitously present throughout the brain, where its distribution in various identified neuronal perikarya and axon tracts is described. Western blot analysis and immunocytochemistry also demonstrated the presence of SNAP-25 in the neural, intermediate and distal lobes of the pituitary gland. Intensity line plots of confocal laser scanning microscope images of isolated melanotropes indicated that SNAP-25 is produced and processed in the rough endoplasmatic reticulum and Golgi apparatus, and is associated with the plasma membrane. Immunoelectron microscopy substantiated the idea that SNAP-25 is present in the plasma membrane but also showed a close association of SNAP-25 with the bounding membrane of peptide-containing secretory granules in both the neurohemal axon terminals in the neural lobe and the endocrine melanotropes in the intermediate lobe. Quantitative Western blotting revealed that adapting Xenopus to a dark background has a clear stimulatory effect on the expression of SNAP-25 in the neural lobe and in the melanotrope cells. This background light intensity-dependent stimulation of SNAP-25 expression was confirmed by the demonstration of increased immunofluorescence recorded by confocal laser scanning microscopy of individual melanotropes of black background-adapted toads. On the basis of this study on Xenopus laevis, we conclude that SNAP-25 (i) plays a substantial role in the secretion of a wide variety of neuronal messengers; (ii) functions in the central nervous system but also in neurohormonal and endocrine systems; (iii) acts at the plasma membrane but possibly also at the membrane of synaptic vesicles and peptide-containing secretory granules; (iv) acts not only locally (as in synapses), but at various sites of the plasma membrane (as in the endocrine melanotrope cell); and (v) can be upregulated in its expression by physiological stimuli that increase the extent of the molecular machinery involved in exocytosis.     

Binding of pemphigus vulgaris IgG to antigens in desmosome core domains excludes immune complexes rather than directly splitting desmosomes

Background Pemphigus vulgaris (PV) is characterized by autoantibodies against desmoglein (Dsg) 3 or both Dsg1 and Dsg3, i.e. desmosomal adhesion molecules. Objectives We examined whether or not PV IgG binding to Dsg3 directly impairs the adhesion of desmosomes. Methods For immunofluorescence microscopy, keratinocytes were first incubated with PV IgG for 30 min in low Ca²⁺ medium, in which no desmosomes were formed, and then for 1 h in high Ca²⁺ medium to generate desmosomes. For immunoelectron microscopy, after a 30‐min incubation with PV IgG in low Ca²⁺ medium, cells were incubated with antihuman IgG with 5‐nm gold particles for 5 min; after washing, cells were further incubated in high Ca²⁺ medium for 1 h. For tracing of PV IgG/Dsg3 immune complexes formed in the desmosomal core domain, cells were first incubated with PV IgG for 5 min to allow PV IgG to bind the desmosomal core domain and were further incubated with PV IgG‐free medium for different times. Results Immunofluorescence microscopy revealed that PV IgG bound in a random‐punctate pattern on the cell surface in low Ca²⁺ medium was translocated to the cell–cell contacts forming a dotted‐linear distribution, suggesting desmosome generation even in the presence of PV IgG. Immunoelectron microscopy revealed that half‐desmosome‐like structures decorated with gold particles in low Ca²⁺ keratinocytes coupled to form desmosomes and gold particles were sandwiched in the desmosomal core domain after Ca²⁺ switch, even though their surfaces were covered with PV IgG/antihuman IgG 5‐nm gold particles. In the tracing experiments, although PV IgG demonstrated a dotted‐linear distribution along the cell–cell contacts colocalized with desmoplakin (DPK) after a 30‐min tracing, it disappeared from cell–cell contacts after a 5‐h tracing, leaving DPK and desmocollin 3. Conclusions These results suggest that the PV IgG/Dsg3 immune complexes are excluded from the desmosomal core domain rather than directly splitting the desmosome.     

N-cadherin expression in palisade nerve endings of rat vellus hairs

Palisade nerve endings (PNs) are mechanoreceptors around vellus hairs of mammals. Each lanceolate nerve ending (LN) of the PN is characterized by a sensory nerve ending symmetrically sandwiched by two processes of type II terminal Schwann cells (tSCIIs). However, the molecular mechanisms underlying the structural organization of the PN are poorly understood. Electron microscopy showed that adherens junctions appeared to adhere to the sensory nerve ending and tSCII processes, so we examined the location of the N-cadherin adhesion system in PNs of rat vellus hairs by using immunoelectron microscopy. N-cadherin localized near both ends of the cell boundary between sensory nerve ending and tSCII processes, which corresponded to the sites of adherens junctions. We further found cadherin-associated proteins, alpha- and beta-catenins, at the linings of adherens junctions. Three-dimensional reconstruction of immunoelectron microscopic serial thin sections showed four linear arrays of N-cadherin arranged longitudinally along the LN beneath the four longitudinal borders of two tSCII processes. In contrast, sensory nerve fibers just proximal to the LNs formed common unmyelinated nerve fibers, in which N-cadherin was located mainly at the mesaxon of type I terminal Schwann cells (tSCIs). These results suggest that the four linear arrays of N-cadherin-mediated junctions adhere the sensory nerve ending and tSCII processes side by side to form the characteristic structure of the LN, and the structural differences between the LNs and the proximal unmyelinated nerve fibers possibly are due to the difference in the pattern of N-cadherin expression between sensory nerve endings and tSCII or tSCI processes.     

Hereditary diffuse leukoencephalopathy with spheroids: ultrastructural and immunoelectron microscopic studies

Hereditary diffuse leukoencephalopathy with spheroids (HDLS) is a rare autosomal dominant disorder characterized by cerebral white matter degeneration with myelin loss and axonal swellings (spheroids) leading to progressive cognitive and motor dysfunction. Histopathology of HDLS has been well characterized, but ultrastructural details are lacking. Here we report ultrastructural and immunoelectron microscopic characterization of spheroids and capillary basal lamina in white matter of HDLS brains. Spheroids had thin or discontinuous or no myelin sheaths. They contained various combinations of aggregated neurofilaments (NF), cytoplasmic organelles, dense bodies, and laminated figures. Aggregated filaments labeled with antibodies to phosphorylated NF (pNF), non-pNF and amyloid precursor protein. The gliotic white matter had many reactive astrocytes, and lipid-laden macrophages with membranous and fingerprint-like bodies. The basal laminas (BL) of many capillaries were dilated, and the enlarged space was heavily deposited with banded collagen type I and III. Some BL had focal thickenings and duplications. Fibronectin, not collagen IV, was found associated with banded collagen. The various types of axonal spheroids and changes in capillary basal lamina have not been emphasized previously. It remains to be determined if they are a reactive process or a primary mechanism of white matter degeneration in HDLS.     

Organotypic keratinocyte coculture using normal human serum: an immunomorphological study at light and electron microscopic levels

Organotypic human skin equivalents of keratinocytes and fibroblasts embedded in collagen matrix have been the subject of studies dealing with various culture conditions. Development of standardized living skin equivalents using defined culture media containing respective supplements can provide important instruments of investigation in skin biology. In addition, tissue engineering has created human skin substitutes for treatment of acute and chronic wounds. In our study, we generate a modified organotypic human skin equivalent using normal human serum instead of fetal calf serum (FCS). This living skin equivalent shows regular stratification of the epidermis and the dermal-epidermal junction zone at the light and electron microscopic level after 1 and 3 weeks of coculture. Indirect immunofluorescence reveals regular expression of differentiation antigens and the major structural proteins collagen IV, laminin 5 and the integrin chains alpha 6 and beta 4 at the dermo-epidermal junction zone. Immunoelectron microscopy demonstrates expression of collagen IV, alpha 6 and beta 4 integrin after 1 and 3 weeks of coculture. This organotypic skin model could be the basis for autologous skin grafting for acute or chronic wounds using autologous serum as well as patients' keratinocytes and fibroblasts, thus minimizing the risk of transmitting infectious agents.     

鼠实验性肝癌超微结构和ras p21免疫电镜观察

目的：动态观察二乙基亚硝胺诱发大鼠肝细胞肝癌过程超微结构和ｒａｓｐ２１免疫电镜改变。方法：对６０只二乙基亚硝胺诱癌的大鼠分期处死,进行电镜及ｒａｓｐ２１免疫电镜观察。结果：早期即见肝细胞内线粒体、粗面内质网、滑面内质网和核、核仁等显示一系列损伤性变化,小肝细胞灶性增生,并向嗜碱细胞和异常肝细胞转化,到诱癌中期以至诱癌晚期,异型肝细胞逐渐向癌细胞转化,并逐渐形成了癌细胞的超微结构特点。ｒａｓｐ２１免疫电镜观察发现,ｐ２１阳性颗粒除沿细胞膜分布外,还可见到内质网上有免疫反应物沉积,甚至在核膜上也观察到略弱的ｐ２１免疫复合物沉积。结论：随诱癌时间的延长,肝细胞逐渐向癌细胞转化,ｒａｓｐ２１阳性颗粒可在癌变细胞内多处分布。