胰岛淀粉样多肽在豚鼠胰腺分布的免疫组织化学研究

本文用免疫组织化学ＡＢＣ法,研究了胰岛淀粉样多肽（Ｉｓｌｅｔａｍｙｌｏｉｄｐｏｌｙｐｅｐｔｉｄｅ,ＩＡＰＰ或称Ａｍｙｌｉｎ）在豚鼠胰脏的分布,并用邻片免疫组织化学双标记法,观察了ＩＡＰＰ与胰岛素（Ｉｎｓｕｌｉｎ,ＩＮＳ）、生长抑素（ＳｏｍａｔｏｓｔａｔｉｎＳＳ）的共存关系。结果显示,豚鼠胰岛内绝大多数细胞都呈ＩＡＰＰ阳性免疫反应,在胰外分泌部的腺泡和导管内也散在分布有ＩＡＰＰ免疫反应阳性细胞。多数ＩＡＰＰ免疫反应阳性的细胞都显示ＩＮＳ免疫反应阳性,胰岛内少数ＩＡＰＰ阳性细胞也呈ＳＳ免疫反应阳性。说明ＩＡＰＰ主要分布在豚鼠的胰岛内．但也少量存在于外分泌部。ＩＡＰＰ主要和ＩＮＳ共存于Ｂ细胞内。但也和ＳＳ共存于Ｄ细胞内,提示ＩＡＰＰ可能通过自分泌途径调节ＩＮＳ和ＳＳ的分泌。     

The effect of pancreatic mesenchyme on the differentiation of endocrine cells from gastric endoderm

To determine whether mesenchyme plays a part in the differentiation of gut endocrine cells, proventricular endoderm from 4- to 5-day chick or quail embryos was associated with mesenchyme from the dorsal pancreatic bud of chick embryos of the same age. The combinations were grown on the chorioallantoic membranes of host chick embryos until they reached a total incubation age of 21 days. Proventricular or pancreatic endoderm of the appropriate age and species reassociated with its own mesenchyme provided the controls. Morphogenesis in the experimental grafts corresponded closely to that in proventricular controls, i.e. the pancreatic mesenchyme supported the development of proventricular glands from proventricular endoderm. Insulin, glucagon and somatostatin cells and cells with pancreatic polypeptide-like immunoreactivity differentiated in the pancreatic controls. The latter three endocrine cell types, together with neurotensin and bombesin/gastrin-releasing polypeptide (GRP) cells, developed in proventricular controls and experimental grafts. The proportions of the major types common to proventriculus and pancreas (somatostatin and glucagon cells) were in general similar when experimental grafts were compared with proventricular controls but different when experimental and pancreatic control grafts were compared. Hence pancreatic mesenchyme did not materially affect the proportions of these three cell types in experimental grafts, induced no specific pancreatic (insulin) cell type and allowed the differentiation of the characteristic proventricular endocrine cell types, neurotensin and bombesin/GRP cells. However, an important finding was a significant reduction in the proportion of bombesin/GRP cells, attributable in part to a decrease in their number and in part to an increase in the numbers of endocrine cells of the other types. This indicates that mesenchyme may well play a part in determining the regional specificity of populations of gut endocrine cells.     

5-羟色胺在豚鼠胰腺分布的免疫组织化学研究

本研究用ABC免疫染色法,结合葡萄糖氧化酶-DAB-硫酸镍铵(Glucose oxidase-DAB-Nickle,GDN)显色技术,在Bouin液固定的常规石蜡切片上,研究了5-羟色胺(5-hydroxytryptamin,5-HT)在豚鼠胰腺内的定位和分布,并用相邻切片免疫双标记,观察了它与胰岛素的共存关系,结果发现,在豚鼠胰腺内,外分泌部均有5-HT免疫反应细胞分布。在胰腺内分泌部(胰岛)5-HT免疫反应细胞分布均匀,大部分胰岛细胞呈阳性5-HT样免疫反应,用相邻薄切片免疫双标记技术证明,胰岛内的5-HT免疫反应细胞主要是B细胞。在胰腺外分泌部,5-HT免疫反应细胞呈单个分散或聚集分布,主要位于腺泡和导管等处,偶见于结缔组织间隔中。本文对研究5-HT在胰腺的生理作用及其机制提供了形态学依据。     

Comparative morphology and biochemistry of pancreatic tissue fragments transplanted into the anterior eye chamber and subcutaneous regions of the rat

The present study was designed to compare the morphological changes occurring in pancreatic tissue fragments transplanted into the anterior eye chamber (AEC) and the subcutaneous (SC) regions of the rat. Pancreatic tissue segments were removed from the tail end of the pancreas of neonatal rats and transplanted into the AEC and SC region of the neck of homologous rats. Five weeks after transplantation, the grafts were removed and processed for light microscopy, immunohistochemistry and radioimmunoassay. In both pancreatic tissue grafts, the acinar cells degenerated completely after transplantation. In contrast to this, insulin-, glucagon-, somatostatin- and pancreatic polypeptide-positive cells and pancreatic ducts survived equally well in both the AEC and SC grafts. The pattern and percentage distribution of insulin-, glucagon-, somatostatin- and PP-producing cells in the AEC and SC grafts was similar to that observed in normal pancreas. However, the percentage distribution of glucagon- and PP-containing cells was significantly (p < 0.03) lower in SC grafts when compared to normal. Radioimmunoassay showed that the AEC and SC pancreatic tissue grafts contained large quantities of insulin and glucagon. However, the insulin content of AEC was slightly but not significantly higher than that of SC grafts. The protein content of pancreatic tissue grafts in these transplantation sites was still significantly (p < 0.05) lower compared to normal. Lymphatic infiltration was also more conspicuous in SC grafts compared to AEC grafts. This infiltration by lymphatic cells was confined only to the endocrine portion of the graft. In conclusion, pancreatic tissue grafts survived in both the AEC and SC regions of rats but the AEC appears to be more conducive to graft survival than the SC region.     

Formation of human islands of Langerhans cells from the epithelium of the acini and ducts]

The contents of endocrine cells in the epithelium of ducts and the number of acino-insular elements in exocrine parenchyma and in pancreatic islets were estimated by means of the dotted method in semithin sections prepared from the pancreas of the human embryos (4--7 months of embryogenesis) and of adult persons (40--50 years of age). Endocrine cell formation was noted in all stages studied in ontogenesis. Quantitative data demonstrated that the epithelium of the ducts is the main source for insulocyte formation. In the pancreas of both human embryos and adult persons, acino-insular transformation participated in the formation of endocrine elements. The data obtained gave a certain evidence on entodermal origin of the pancreatic islets in the human pancreas.     

大鼠胰腺嗜铬颗粒素A分布的免疫组织化学研究

本研究用ＡＢＣ免疫组织化学方法,在Ｂｏｕｉｎ液固定的常规石蜡切片上,观察了啥铬颗粒素Ａ在大鼠胰腺内分泌细胞内的定位和分布,并用相邻切片双标记法,观察了它与胰高血糖素、胰岛素、生长抑素的共存关系。结果发现,大鼠胰腺嗜铬颗粒素Ａ样免疫反应细胞主要分布于胰岛的周边部,胰腺外分泌部的导管和腺泡等处均未见ＣｇＡ祥物质存在。用相邻薄切片免疫显色技术证明,大鼠胰腺中ＣｇＡ样物质与胰高血糖素共存。结果提示,ＣｇＡ可能是胰腺内分泌细胞的一个新的标志物,在胰腺功能调节上发挥着重要作用。     

Impaired differentiation of endocrine and exocrine cells of the pancreas in transgenic mouse expressing the truncated type II activin receptor

Activin A is expressed in endocrine precursor cells of the fetal pancreatic anlage. To determine the physiological significance of activins in the pancreas, a transgenic mouse line expressing the truncated type II activin receptor under the control of beta-actin promoter was developed. Histological analyses of the pancreas revealed that the pancreatic islets of the transgenic mouse were small in size and were located mainly along the pancreatic ducts. Immunoreactive insulin was detected in islets, some acinar cells, and in some epithelial cells in the duct. In addition, there were abnormal endocrine cells outside the islets. The shape and the size of the endocrine cells varied and some of them were larger than islets. These cells expressed immunoreactive insulin and glucagon. In the exocrine portion, there were morphologically abnormal exocrine cells, which did not form a typical acinar structure. The cells lacked spatial polarity characteristics of acinar cells but expressed immunoreactive amylase, which was distributed diffusely in the cytoplasm. Plasma glucose concentration was normal in the transgenic mouse before and after the administration of glucose. The insulin content of the pancreas in transgenic and normal mice was nearly identical. These results suggest that activins or related ligands regulate the differentiation of the pancreatic endocrine and exocrine cells.     

Development of the endocrine pancreas during larval phases of Rana temporaria

Summary The pancreatic endocrine component was studied at different stages of development in the tadpoles of Rana temporaria. The material was embedded in Epon, and serial semithin and thin sections were made in order to correlate ultrastructural features and tinctorial traits of the endocrine cells. Serial semithin sections were also stained with the peroxidase-antiperoxidase immunocytochemical method and with silver impregnations for argyrophilia and argentaffinity. In early larvae (legless tadpoles), A and B cells are present. Both can be found within ducts and exocrine tissue or, more frequently, in cellular clusters among the ducts and acini. These primitive islets are solid structures, surrounded but not penetrated by capillaries. Mitoses were observed in A and B cells. In the following phase (tadpoles with hindlegs), D and pancreatic polypeptide-immunoreactive cells are also present, as well as numerous endocrine cells scattered among exocrine tissue. There is also a change in the vascular-insular pattern: capillaries not only surround but also penetrate the endocrine group. The structure of the endocrine pancreas in older tadpoles is similar. Tinctorial traits and ultrastructural features of endocrine cells are described, and the origin of primitive islets is discussed.     

Pancreatic duct occlusion with Ethibloc. An experimental study in juvenile pigs

In the transplantation of vascularized pancreatic grafts severe problems are related to the exocclusion is an improved alternative to duct ligation in producing atrophy of the exocrine pancreas while leaving the endocrine pancreas intact. Fractional growth rate in duct-ligated and duct-occluded animals was reduced to 1/3 - 1/4 of that of sham operated controls. Fasting blood glucose, fasting insulin, sum of blood glucose and glucose elimination rate during an intravenous glucose tolerance test remained normal in the duct-ligated and Ethibloc-occluded animals. There was a diminished insulin response to a maximal glucose load. In spite of this, the glucose tolerance remained virtually normal. The volume of the pancreas was reduced to 1/3 of its normal size after both experimental procedures. Histologically, the islets appeared to remain normal, while the exocrine portion of the gland was replaced by fibrous tissue. No traces of the active compound, Ethibloc, remained after 4 weeks. This study shows that pancreatic duct occlusion with Ethibloc results in impairment of endocrine function. Consequently, Ethibloc duct occlusion does not seem to be a superior alternative to other methods of producing exocrine atrophy in organs intended for transplantation.     

An immunocytochemical study of fetal cells at the maternal-placental interface using monoclonal antibodies to keratins,vimentin and desmin

Summary Thioredoxin and thioredoxin reductase (NADPH-oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) have been proposed to be involved in several thioldependent reduction-oxidation reactions in cells. Both proteins have been immunohistochemically demonstrated in the periphery of the cytoplasm and in cytoplasmic granules of acinar and islet cells in mouse pancreas. In animals fed ad libitum, the staining for thioredoxin was more intense in the exocrine acinar cells than in the islet cells, whereas that for thioredoxin reductase was more intense in the endocrine than in the exocrine pancreas. In the islets of fed mice all endocrine cell types showed about the same staining intensity for thioredoxin, while thioredoxin reductase was greatly enriched in the somatostatin-containing D cells. Starvation overnight caused an increased staining for both proteins in the acinar cells as well as in the islets. Under conditions of starvation, thioredoxin reductase, in contrast to thioredoxin, appeared to increase preferentially in the islet B cells, as compared with the D cells. Cysteamine treatment reduced the staining for somatostatin and for thioredoxin reductase in the D cells without any obvious effect on the other pancreatic cells. The results are compatible with a role for thioredoxin and thioredoxin reductase in secretion.     

Evolutionary conserved role of ptf1a in the specification of exocrine pancreatic fates

We have characterized and mapped the zebrafish ptf1a gene, analyzed its embryonic expression, and studied its role in pancreas development. In situ hybridization experiments show that from the 12-somite stage to 48 hpf, ptf1a is dynamically expressed in the spinal cord, hindbrain, cerebellum, retina, and pancreas of zebrafish embryos. Within the endoderm, ptf1a is initially expressed at 32 hpf in the ventral portion of the pdx1 expression domain; ptf1a is expressed in a subset of cells located on the left side of the embryo posteriorly to the liver primordium and anteriorly to the endocrine islet that arises from the posterodorsal pancreatic anlage. Then the ptf1a expression domain buds giving rise to the anteroventral pancreatic anlage that grows posteriorly to eventually engulf the endocrine islet. By 72 hpf, ptf1a continues to be expressed in the exocrine compartment derived from the anteroventral anlage. Morpholino-induced ptf1a loss of function suppresses the expression of the exocrine markers, while the endocrine markers in the islet are unaffected. In mind bomb (mib) mutants, in which delta-mediated notch signalling is defective [Dev. Cell 4 (2003) 67], ptf1a is normally expressed. In addition, the slow-muscle-omitted (smu) mutants that lack expression of endocrine markers because of a defective hedgehog signalling [Curr. Biol. 11(2001) 1358] exhibit normal levels of ptf1a. This indicates that hedgehog signaling plays a different genetic role in the specification of the anteroventral (mostly exocrine) and posterodorsal (endocrine) pancreatic anlagen.     

Conserved origin of the ventral pancreas in chicken

To determine the origin of the ventral pancreas, a fate map of the ventral pancreas was constructed using DiI crystal or CM-DiI to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. First, the region lateral to the 7- to 9-somite level, which has been reported to contribute to the ventral pancreas, was shown to contribute mainly to the intestine or the dorsal pancreas. At the 10 somite stage (ss), the ventral pre-pancreatic cells reside laterally at the 2-somite level, at the lateral boarder of the somite. At this stage, however, the fate of these cells has not yet segregated and they contribute to the ventral pancreas and to the intestine or bile duct. The ventral pancreas fate segregated at the 17 ss; the cells residing at the somite boarder at the 4-somite level at the 17 ss were revealed to contribute to the ventral pancreas. Interestingly, the dorsal and the ventral pancreatic buds are different in both origin and function. These two pancreatic buds begin to fuse at day 7 (HH 30) of embryonic development. However, whereas the dorsal pancreas gives rise to both Insulin-expressing endocrine and Amylase-expressing exocrine cells, the ventral pancreas gives rise to Amylase-expressing exocrine cells, but not insulin-expressing endocrine cells before day 7 (HH 30) of embryonic development.     

Evidence for Epithelial�CMesenchymal Transition in Adult Human Pancreatic Exocrine Cells

It has been shown that adult pancreatic ductal cells can dedifferentiate and act as pancreatic progenitors. Dedifferentiation of epithelial cells is often associated with the epithelial–mesenchymal transition (EMT). In this study, we investigated the occurrence of EMT in adult human exocrine pancreatic cells both in vitro and in vivo. Cells of exocrine fraction isolated from the pancreas of brain-dead donors were first cultured in suspension for eight days. This led to the formation of spheroids, composed of a principal population of cells with duct-like phenotype. When cultivated in tissue culture-treated flasks, spheroid cells exhibited a proliferative capacity and coexpressed epithelial (cytokeratin7 and cytokeratin19) and mesenchymal (vimentin and α-smooth muscle actin) markers as well as marker of progenitor pancreatic cells (pancreatic duodenal homeobox factor-1) and surface markers of mesenchymal stem cells. The switch from E-cadherin to N-cadherin associated with Snail1 expression suggested that these cells underwent EMT. In addition, we showed coexpression of epithelial and mesenchymal markers in ductal cells of one normal adult pancreas and three type 2 diabetic pancreases. Some of the vimentin-positive cells were found to coexpress glucagon or amylase. These results point to the occurrence of EMT, which may take place on dedifferentiation of ductal cells during the regeneration or renewal of human pancreatic tissues. (J Histochem Cytochem 58:807–823, 2010)     

Distinct delta and jagged genes control sequential segregation of pancreatic cell types from precursor pools in zebrafish

The different cell types of the vertebrate pancreas arise asynchronously during organogenesis. Beta-cells producing insulin, alpha-cells producing glucagon, and exocrine cells secreting digestive enzymes differentiate sequentially from a common primordium. Notch signaling has been shown to be a major mechanism controlling these cell-fate choices. So far, the pleiotropy of Delta and Jagged/Serrate genes has hindered the evaluation of the roles of specific Notch ligands, as the phenotypes of knock-out mice are lethal before complete pancreas differentiation. Analyses of gene expression and experimental manipulations of zebrafish embryos allowed us to determine individual contributions of Notch ligands to pancreas development. We have found that temporally distinct phases of both endocrine and exocrine cell type specification are controlled by different delta and jagged genes. Specifically, deltaA knock-down embryos lack alpha cells, similarly to mib (Delta ubiquitin ligase) mutants and embryos treated with DAPT, a gamma secretase inhibitor able to block Notch signaling. Conversely, jagged1b morphants develop an excess of alpha-cells. Moreover, the pancreas of jagged2 knock-down embryos has a decreased ratio of exocrine-to-endocrine compartments. Finally, overexpression of Notch1a-intracellular-domain in the whole pancreas primordium or specifically in beta-cells helped us to refine a model of pancreas differentiation in which cells exit the precursor state at defined stages to form the pancreatic cell lineages, and, by a feedback mediated by different Notch ligands, limit the number of other cells that can leave the precursor state.     

The distribution and localization of the monoclonal antibody-defined antigen 19-9 (CA19-9) in chronic pancreatitis and pancreatic carcinoma. An immunohistochemical study

The carbohydrate antigen 19-9 (CA19-9) is considered to be of great importance in the diagnosis, differential diagnosis and follow-up of human pancreatic carcinoma. CA19-9 antigen has been isolated and characterized as the oligosaccharide sialylazed lacto-N-fucopentaose II and a monoclonal antibody against CA19-9 is commercially available. In this immunochemical study we have examined the localisation and distribution of monoclonal anti-CA19-9 in pancreatic tissue obtained from 20 patients with a normal pancreas (lacking pancreatic tumour or evidence of inflammation), from 50 patients with chronic pancreatitis and from 50 patients with pancreatic carcinomas of various types. In the normal pancreas (free from tumour or inflammation) we found anti-CA19-9 to be localized in the branches of the pancreatic ducts with discontinuities predominantly at the apical surfaces of the lining epithelium. In chronic pancreatitis a continuous positive reaction was found in the small, medium and large ramifications of the pancreatic ducts. In ductal epithelium exhibiting mucoid transformation, a mosaic-like, discontinuous positive reaction was found, whereas in epithelium showing pseudopapillary and papillary hyperplasia a uniform positive reaction was obtained. Multilayered epithelium ("squamous metaplasia") was negative. The fluid content of any cysts present and the tubular accumulations found in chronic pancreatitis showed a positive reaction. The reaction in chronic pancreatitis differed from that in normal pancreas in its distribution but not in its intensity. All carcinomas of the exocrine pancreas showed intensely positive reaction in a very varied distribution whereas the anaplastic carcinomas gave a negative reaction. Whilst in chronic pancreatitis the binding of anti-CA19-9 was unimpressive and strictly localized, in exocrine pancreatic carcinomas binding was and strictly localized, in exocrine pancreatic carcinomas binding was very marked and diffuse in distribution. From this we conclude that malignant cells display a greater number of CA19-9 epitopes than cells in chronic pancreatitis. The difference can only be regarded as quantitative, since the immunohistochemical reaction does not allow qualitative discrimination between chronic pancreatitis and pancreatic carcinoma; CA19-9 should not be therefore termed a "tumour marker". The glycoprotein nature of CA19-9 was confirmed by sialidase and chemical desialylation.     

Formation of the digestive system in zebrafish. II. Pancreas morphogenesis

Recent studies have suggested that the zebrafish pancreas develops from a single pancreatic anlage, located on the dorsal aspect of the developing gut. However, using a transgenic zebrafish line that expresses GFP throughout the endoderm, we report that, in fact, two pancreatic anlagen join to form the pancreas. One anlage is located on the dorsal aspect of the developing gut and is present by 24 h postfertilization (hpf), the second anlage is located on the ventral aspect of the developing gut in a position anterior to the dorsal anlage and is present by 40 hpf. These two buds merge by 52 hpf to form the pancreas. Using heart and soul mutant embryos, in which the pancreatic anlagen most often do not fuse, we show that the posterior bud generates only endocrine tissue, while the anterior bud gives rise to the pancreatic duct and exocrine cells. Interestingly, at later stages, the anterior bud also gives rise to a small number of endocrine cells usually present near the pancreatic duct. Altogether, these studies show that in zebrafish, as in the other model systems analyzed to date, the pancreas arises from multiple buds. To analyze whether other features of pancreas development are conserved and investigate the influence of surrounding tissues on pancreas development, we examined the role of the vasculature in this process. Contrary to reports in other model systems, we find that, although vascular endothelium is in contact with the posterior bud throughout pancreas development, its absence in cloche mutant embryos does not appear to affect the early morphogenesis or differentiation of the pancreas.     

The ductal origin of structural and functional heterogeneity between pancreatic islets

Islets form in the pancreas after the first endocrine cells have arisen as either single cells or small cell clusters in the epithelial cords. These cords constitute the developing pancreas in one of its earliest recognizable stages. Islet formation begins at the time the cords transform into a branching ductal system, continues while the ductal system expands, and finally stops before the exocrine tissue of ducts and acini reaches its final expansion. Thus, islets continuously arise from founder cells located in the branching and ramifying ducts. Islets arising from proximal duct cells locate between the exocrine lobules, develop strong autonomic and sensory innervations, and pass their blood to efferent veins (insulo-venous efferent system). Islets arising from cells of more distal ducts locate within the exocrine lobules, respond to nerve impulses ending at neighbouring blood vessels, and pass their blood to the surrounding acini (insulo-acinar portal system). Consequently, the section of the ductal system from which an islet arises determines to a large extent its future neighbouring tissue, architecture, properties, and functions. We note that islets interlobular in position are frequently found in rodents (rats and mice), whereas intralobularly-located, peripheral duct islets prevail in humans and cattle. Also, we expound on bovine foetal Laguesse islets as a prominent foetal type of type 1 interlobular neuro-insular complexes, similar to neuro-insular associations frequently found in rodents. Finally, we consider the probable physiological and pathophysiological implications of the different islet positions within and between species.