报告一种同时显示肥大细胞、血管、神经等组织成分的新染色方法

目的为了同时观察组织切片内肥大细胞、血管、神经等组织成分彼此间的形态学关系和联系。方法取大鼠后肢皮肤组织,经恒冷箱切片机切片,然后将组织切片先后经亚铁氰化铜法示乙酰胆碱酯酶和甲苯胺蓝染液染色。结果可同时在组织切片观察到肥大细胞、血管和乙酰胆碱酯酶阳性神经纤维等,以及它们彼此形成的接触联系。结论实验建立的这种新染色方法是显示组织内肥大细胞与血管和神经发生形态学联系的有效方法。     

脱钙对鼠颅骨标本中肥大细胞组织化学与免疫组织化学染色的影响

目的:探讨脱钙对鼠颅骨标本中肥大细胞组织化学与免疫组织化学染色影响.方法:用不同脱钙液处理小鼠颅骨标本,组织切片后进行苏木素一伊红染色、甲苯胺蓝染色、醛品红染色以及免疫组织化学染色.结果:经过不同脱钙液处理后的颅骨组织切片组织结构保存完好,肥大细胞的组织化学染色(甲苯胺蓝和醛品红染色),及肥大细胞中胰蛋白酶的免疫组织化学染色清晰.结论:不同脱钙液(8%盐酸脱钙液、EDTA脱钙液、混合酸脱钙液)处理不影响鼻腔粘膜中肥大细胞的组织化学和免疫组织化学染色.     

新鲜组织肥大细胞快速染色方法的探讨与应用

目的探讨如何在组织内更好地显示肥大细胞。方法取大鼠后肢皮肤组织,经恒冷箱切片机切片,然后将组织切片直接入1%甲苯胺蓝染液进行染色。结果在新鲜组织切片内可见到较多充满蓝紫色颗粒的肥大细胞和它们分泌的蓝紫色颗粒。结论新鲜组织直接进行甲苯胺蓝染色可显示较多的肥大细胞。     

牛蛙肥大细胞的组织化学与形态学

目的鉴定牛蛙组织中肥大细胞的存在。方法用于肥大细胞研究的一些常规组织化学技术与形态学方法。结果牛蛙的舌、肠、肠系膜和脾中肥大细胞数量较多,少量也见于神经、心、肾、肝和皮肤等多种组织中。肥大细胞有沿血管周和神经分布的倾向。脾脏中的肥大细胞形状比较一致,呈圆形或卵圆形,而在其它部位的肥大细胞则形态多样。Bouin氏液及Carnoy氏液是牛蛙肥大细胞优良的固定液。然而,与哺乳动物的黏膜肥大细胞相似的是,中性缓冲福尔马林(NBF)固定显著的阻断了牛蛙肠黏膜肥大细胞(MMC)的染色。有趣的是,甲苯胺蓝是牛蛙肥大细胞的最佳染料,它比阿尔新蓝能很好地显示牛蛙的肥大细胞。透射电镜下证实,牛蛙肥大细胞中含有大量特征性的胞浆颗粒。肥大细胞靠近雪旺氏细胞,并可见于神经束膜间,甚至以其突起与神经束膜相连。结论通过组织化学与形态学研究证实了牛蛙组织中肥大细胞的存在,再次证实肥大细胞与外周神经之间存在密切的解剖学关系。     

家兔脊神经节内肥大细胞与肽能神经关系的观察

探讨脊神经节内肥大细胞与肽能神经的关系,采用常规组织学染色和免疫组织化学方法对家兔脊神经节内肥大细胞和P物质免疫阳性反应进行观察。结果显示：在P物质免疫反应阳性的神经元和神经纤维周围散在着肥大细胞,表明,脊神经节内,肥大细胞与肽能神经存在着组织形态学上的构筑关系。     

改良的肥大细胞甲苯胺蓝染色法

目的:本研究旨在运用一种改良的甲苯胺蓝染色方法鉴定体外分离培养的肥大细胞形态,为肥大细胞研究提供简便的检测方法。方法:体外诱导分化培养小鼠骨髓来源的肥大细胞,4周后收集细胞、固定、染色。比较不同固定温度及时间对甲苯胺蓝染色效果的影响,确定最佳条件。结果:SCF和IL-3体外培养诱导出小鼠骨髓来源肥大细胞。肥大细胞用改良的甲苯胺蓝染色后细胞着色效果较好,细胞多呈圆形或椭圆形且胞膜较完整,胞浆中充满大量的紫红色颗粒。结论:本研究成功运用一种适用于体外培养小鼠骨髓源肥大细胞的甲苯胺蓝染色法,并发现肥大细胞在37℃充分固定后进行染色,可以降低肥大细胞发生脱颗粒。此操作方法稳定性好、简便,适用于体外培养的肥大细胞形态学观察。     

肥大细胞的组织化学与超微结构异质性

肥大细胞(mast cell,MC)是一种重要的免疫细胞,分为结缔组织肥大细胞(connective tissue mast cell,CTMC)和黏膜肥大细胞(mucosal mast cell,MMC)两大类。肥大细胞具有异质性,即肥大细胞在不同种属或同一种种属的不同个体、甚至同一种个体的不同组织器官中存在着形态学、分布、颗粒化学成分、染色特性及超微结构和功能等方面的差异性。近些年,人们围绕着肥大细胞的异质性进行了一系列生物学研究,并取得了一定进展,但对异质性的机制认识尚不清楚。深入的讨论、研究与比较仍然很必要。现对肥大细胞的亚群、形态与分布、着染性与免疫组化、超微结构等的异质性研究进展作一简要综述。     

实验动物呼吸系统主要器官比较组织学研究

目的比较实验动物呼吸系统主要器官的组织学特征,为制定实验动物病理检测标准、以及毒理学、新药安全性评价提供依据。方法选取实验动物质量国家检测标准检测合格的恒河猴30只、昆明小鼠20只、SD大鼠20只、日本大耳白兔18只、比格犬16只、树鼩20只。除昆明小鼠采用颈椎脱臼致死外,其余动物麻醉后放血处死和病理解剖,对气管、肺脏进行病理大体检查和取材,常规病理制片,进行HE染色、特殊染色和免疫组化染色,显微镜下观察气管、肺脏的组织结构和细胞结构异同。结果 （1）实验动物气管上皮杯状细胞有差异：恒河猴、比格犬、日本大耳白兔杯状细胞较多,大鼠、小鼠、树鼩则较少或无。上皮分泌的黏液类型以中性黏液为主,比格犬杯状细胞分泌的黏液类型有中性黏液和酸性黏液。（2）实验动物黏膜下腺泡分布有差异：比格犬黏膜下层的腺泡最多,恒河猴、大鼠、小鼠、树鼩腺泡数量偏少,日本大耳白兔黏膜下层的混合腺泡最少。（3）实验动物的肺内支气管分支有差异：比格犬、恒河猴、日本大耳白兔由叶支气管、段支气管、小支气管、细支气管、终末细支气管和呼吸性细支气管组成,树鼩、大鼠、小鼠只由细支气管、终末细支气管和呼吸性细支气管组成。（4）实验动物细支气管组织结构有差异：恒河猴、比格犬的细支气管平滑肌为完整环形平滑肌层,没有缺失,而大鼠、小鼠、树鼩及日本大耳白兔的细支气管平滑肌薄或缺失。恒河猴、树鼩、大鼠细支气管有少量杯状细胞,其余实验动物均无杯状细胞。（5）实验动物Clara细胞形态有差异：比格犬Clara细胞呈立方形,其余动物呈柱状。结论实验动物呼吸系统组织结构的质是相同的,差异在于量的不同。研究人员在制定病理学检测标准、实验研究、药物安全性评价时应予充分考虑。     

肥大细胞用甲苯胺蓝染色之体会

肥大细胞是存在于多种组织内的一类细胞 ,并因所在组织器官的不同而具有不同的功能。肥大细胞是抗感染免疫的第一线细胞 ,在疾病过程中具有重要作用 ,因此 ,研究肥大细胞的生物学性质具有重要的理论意义和广泛的应用价值。常用的肥大细胞染色方法有甲苯胺蓝染色法、阿尔辛蓝—番红染色法等。肥大细胞具有复杂的异质性 ,因此上述方法不仅难以全面反映其生物学特性 ,而且有时也难客观地反映其在组织中的分布。我们用改良的甲苯胺蓝染色法对食管、肠、子宫、乳腺等器官中的肥大细胞进行染色时得到了一点体会 ,供参考。甲苯胺蓝改良染色法 :根据…     

间接免疫过氧化物酶技术鉴定猪和牛的肥大细胞

用小鼠抗人肥大细胞类胰蛋白酶单克隆抗体ＡＡ１,ＡＡ３及ＡＡ５的间接免疫过氧化物酶技术对经Ｃａｒｎｏｙ液或中性缓冲福尔马林固定的猪和犊牛空肠,舌及胸腺的石蜡切片进行了免疫染色。对猪和牛的肥大细胞特异性免疫染色与常规的组织化学染色的结果进行了比较。     

Eosinophils and mast cell homogeneity of the guinea pig eyelid skin, conjunctiva, and ileum

Mast cell heterogeneity has been described on the basis of differential staining reactions, light microscopic morphology, anatomic location, degranulation after polyamines, biochemical contents, growth requirements, and reactions to lymphokines. We have demonstrated typical "connective-tissue mast cells" by using anatomic criteria, histological staining reactions, electron microscopy, and reaction to compound 48/80 in the guinea pig conjunctiva, eyelid skin, and ileum. A second, much larger population of cells in the ileal mucosa and the conjunctiva, and rarely in the eyelid skin stained reddish-blue with acid toluidine blue in tissue fixed in ethanol-acetate-lead subacetate (BLA) and with alkaline Giemsa in formaldehyde-fixed tissue, did not stain with ethanolic or acid toluidine blue in formaldehyde-fixed tissue or with alkaline Giemsa in BLA-fixed tissue, and did not degranulate after 48/80 treatment. These are features of the rat intestinal "mucosal mast cells"; however, ultrastructural and light microscopic studies with the orcein Giemsa stain demonstrated these cells in the guinea pig to be eosinophils. Tissue culture, biochemical, and immunological studies indicate the existence of a second type of mast cell (bone-marrow-derived mast cell), ultrastructurally almost indistinguishable from the connective tissue mast cell. Our studies demonstrate only one mast cell type in the guinea pig and support the contention that other forms of mast cells are immature forms or variants of the connective-tissue mast cell.     

Characteristics on staining of leukocytes in experimental animals]

The staining characteristics of the peripheral blood cells from mouse, rat, guinea pig, rabbit, dog, marmoset and monkey were studied. In marmoset, it is easy to distinguish neutrophils from eosinophils by using the phosphate-buffered solution of pH 5 or 6. It was found in the special staining methods that neutrophil granules showed intense peroxidase and Sudan black B reactions in marmoset in comparison with those in the other species of experimental animals. Neutrophil granules rabbit was, however, intensely stained with esterase and acid phosphatase.     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

BN大鼠和豚鼠对卵清白蛋白致主动过敏反应的免疫学特性比较

目的 比较BN大鼠和豚鼠对卵清白蛋白(OVA)致敏前后机体免疫学特性的变化.方法 BN大鼠和豚鼠分别用OVA(每只1 mg)隔日致敏(i.p.),共5次;于末次致敏第10天以OVA(每只2 mg)激发致敏(i.v.);分别设正常对照组和OVA致敏组.于激发致敏后1h处死动物,分离腹腔肥大细胞、脾脏和骨髓,并制备脾脏和骨髓淋巴细胞.以annexin-V作为标志检测肥大细胞活性,同时以Fluo-3/AM标记胞内钙离子,检测钙离子水平;以PHA和LPS作为有丝分裂原,分别检测脾脏和骨髓T、B淋巴细胞活性.结果 ①致敏BN大鼠和豚鼠脾脏及骨髓T、B淋巴细胞活性均升高,其中骨髓淋巴细胞活性BN大鼠显著高于豚鼠,脾脏淋巴细胞活性两种属间差异无显著性;②致敏后,腹腔肥大细胞活性两种属间差异无显著性,但BN大鼠致敏后是致敏前的6倍,豚鼠是3倍;③肥大细胞内钙离子水平两种属致敏后均升高,豚鼠致敏前后钙离子水平具有统计学意义.结论 OVA致敏后,BN大鼠骨髓淋巴细胞活性明显高于豚鼠,豚鼠肥大细胞内钙离子较BN大鼠升高明显,肥大细胞活性两者无明显差异.因此,在实验中可以根据两种属在过敏反应中的特点以及具体的实验要求选择动物模型.     

Formation of mast cell colonies in methylcellulose by mouse peritoneal cells and differentiation of these cloned cells in both the skin and the gastric mucosa of W/Wv mice: evidence that a common precursor can give rise to both "connective tissue-type" and "mucosal" mast cells

We investigated the issue of mast cell heterogeneity by cloning mast cell colonies from peritoneal cells in methylcellulose, injecting the cloned cells into the skin and stomach of mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice, and staining the mast cells that developed in these sites with Berberine sulfate, a fluorescent dye that identifies heparin-containing mast cells. When peritoneal cells of nontreated WBB6F1-+/+ mice were plated in methylcellulose containing pokeweed mitogen-stimulated spleen cell conditioned medium, pure mast cell colonies developed. In contrast, the peritoneal cavity of genetically mast cell-deficient WBB6F1-W/Wv mice lacked the progenitor cells that made mast-cell colonies. The clonal nature of the mast cell colonies was determined by using the giant granules of C57BL/6-bgJ/bgJ mice as a marker: even when mixture of peritoneal cells of C57BL/6-bgJ/bgJ mice and C57BL/6-+/+ mice were plated, all of the resulting colonies consisted of either bgJ/bgJ-type mast cells alone or +/+-type mast cells alone. Individual mast c 11 colonies of WBB6F1-+/+ mouse origin were divided into two parts; one part was directly injected into the wall of the glandular stomach of a WBB6F1-W/Wv mouse, and another part was injected into the skin of the same W/Wv mouse. Injections of 14 of 46 such colonies resulted in development of mast cells in both the "connective tissues" (skin or stomach muscle or both) and the stomach mucosa. Mast cells in the connective tissues were stained with Berberine-sulfate, indicating that they contained heparin, whereas mast cells in the stomach mucosa were not. These results suggest that a single precursor cell can give rise to both "connective tissue-type" and "mucosal" mast cells.     

Use of the polymerase chain reaction for homology probing of butyrylcholinesterase from several vertebrates

Genomic blots from man, monkey, cow, sheep, pig, rabbit, dog, rat, mouse, guinea pig, and chicken DNA were hybridized with probes derived from the four exons of the human butyrylcholinesterase gene (BCHE) (Arpagaus, M., Kott, M., Vatsis, K. P., Bartels, C. F., La Du, B. N., and Lockridge, O. (1990) Biochemistry 29, 124-131). Results showed that the BCHE gene was present in a single copy in the genome of all these vertebrates. The polymerase chain reaction was used to amplify genomic DNA from these animals with oligonucleotides derived from the human BCHE coding sequence. The amplified segment contained 423 bp of BCHE sequence including the active site serine of the enzyme (amino acid 198) and a component of the anionic site, aspartate 70. Amplification was successful for monkey, pig, cow, dog, sheep, and rabbit DNA, but unsuccessful for rat, guinea pig, mouse, and chicken DNA. Amplified segments were cloned in M13 and sequenced. The mouse sequence was obtained by sequencing a genomic clone. The highest identity of the human amino acid sequence was found with monkey (100%) and the lowest with mouse (91.5%). The sequence around the active site serine 198, Phe-Gly-Glu-Ser-Ala-Gly-Ala, was conserved in all eight animals as was the anionic site component, aspartate 70. A phylogenetic tree of mammalian butyrylcholinesterases was constructed using the partial BCHE sequences.     

On the nature of the metachromatic cells of pancreatic islets

Summary Islet cells with cytoplasmic granules metachromatically stained by toluidine blue are found in the pancreas of dog, cat, pig, rabbit, teleostian fish, monkey and man, but not in the rat, mouse and guinea pig; dubious results are obtained on duck and ox islets. These cells do not react with the staining methods for the demonstration of A- and B-cells; conversely, they are silver-impregnated by a modification of Davenport's method, and, at least in dog islets, can be readily identified with dark-field microscopy and stained blue with the Mallory-Heidenhain trichrome stain. No obvious changes of this cell type are found either in synthalintreated dogs or in diabetic men.The Authors suggest that the islet argyrophil-metachromatic cells (A₁-cells of Swedish Authors) are identical with D-cells and very likely represent a morphologically and functionally indipendent cell type.     

Effect of anti-lymphotoxin on cell-mediated cytotoxicity. Evidence for two pathways, one involving lymphotoxin and the other requiring intimate contact between the plasma membranes of killer and target cells.

A rabbit anti-lymphotoxin serum produced against partially purified, antigeninduced, guinea pig lymphotoxin, was used to study the role of lymphotoxin in lymphocyte-mediated cytotoxicity in vitro. The anti-lymphotoxin serum inhibited cytolysis resulting from incubation of ovalbumin-immune guinea pig spleen cells with either mouse (P815 mastocytoma) or guinea pig (line 10 hepatoma) target cells in the presence of soluble ovalbumin. The antiserum also inhibited the cytolysis of ovalbumin-coupled target cells by ovalbumin-immune guinea pig spleen cells. In contrast, the anti-lymphotoxin serum did not inhibit: (a) the lysis of line 10 (strain 2) hepatoma cells by spleen cells from alloimmunized Hartley or strain 13 animals; (b) the lysis of line 10 hepatoma cells by spleen cells from tumor-bearing syngeneic animals; or (c) the lysis of P815-mastocytoma cells by spleen cells from P815-immune guinea pigs. These results support the hypothesis that there are at least two distinct pathways by which immune lymphocytes can destroy target cells in vitro, one which involves secretion of a nonspecific soluble factor, i.e., lymphotoxin, and another which probably requires intimate contact between the plasma membranes of the target and killer cells.     

Histochemistry and morphology of porcine mast cells

Summary Mast cells have been described extensively in rodents and humans but not in pigs, and the objective of this study was to characterize porcine mast cells by histochemistry and electron microscopy. Carnoy's fluid proved to be a good fixative but fixation with neutral buffered formalin blocked staining of most mast cells. Alcian Blue stained more mast cells than did Toluidine Blue (pH 0.5), although Alcian Blue also stained goblet cells. In pigs, unlike rodents, the Alcian Blue method did not distinguish between mast cells in the intestinal mucosa and those in the connective tissue of the intestinal submucosa, tongue and skin. Mast cells were significantly larger in adult pigs than in piglets; in adult pigs and piglets, mast cells in the intestinal mucosa were significantly larger than those in submucosal connective tissue, and they were more varied in shape in piglets and adults. Granules in mast cells in the intestinal mucosa stained less intensely than those in mast cells in connective tissue of tongue, skin and intestinal submucosa. Mast cells in the connective tissue of the tongue, skin and intestinal submucosa fluoresced strongly when stained with berberine sulphate or with a mixture of berberine sulphate and Acridine Orange, but mast cells in the intestinal mucosa did not. All mast cells reacted positively in an enzyme-histochemical method previously used to detect human tryptase but not in a method previously used to detect human chymase. Mast cells in the medulla of thymus stained similarly to mast cells in the intestinal mucosa. Ultrastructural differences between mast cells were not detected.     

Activation and inhibition of mast cells degranulation affect their morphometric parameters

Activation of mast cells, the key cells of allergic inflammation, causes typical morphological changes associated with an increase in volume, that is a function of area and perimeter. The purpose of this study was to evaluate the effect of mast cell activation to degranulate, carried out by the secretagogue Compound 48/80, and of inhibition of this activation carried out by Nedocromil sodium, a mast cell stabilizing drug, on mast cell area, perimeter and shape factor by a computerized image analyzer. Mast cells were isolated and purified by peritoneal lavage of rats (purity >98%) and co-cultured with mouse 3T3 fibroblasts to which they adhere. Cultures were incubated for 10 min at 37 degrees C with culture medium alone (Enriched Medium) or Enriched Medium containing either Nedocromil (10(-4) M) or Compound 48/80 (0.3 microg/ml) or Compound 48/80 and Nedocromil (0.3 microg/ml and 10(-4) M respectively). Supernatants were then assessed for histamine release, as a marker of mast cell activation and the cell monolayers were fixed and stained with an alcoholic-acidic toluidine blue solution and examined with a computerized image analyzer connected with a light microscope. Mast cells incubated in Enriched Medium or Nedocromil possessed similar morphometric parameters. Mast cells activated with Compound 48/80 (70% histamine release) had a significant increase in area and perimeter and a decrease in shape factor in comparison to mast cells in Enriched Medium alone. Simultaneous incubation of mast cells with Compound 48/80 and Nedocromil significantly inhibited their histamine release (36% histamine release) and the increase in area and perimeter, but did not affect significantly their shape factor, in comparison with mast cells incubated with Compound 48/80 alone. These data clearly show that there is a relationship between mast cell activation, consequent histamine release and changes in cell area, perimeter and shape factor and that Nedocromil not only inhibits mast cell histamine release but also the activation induced morphometric changes in mast cells.