资木瓜中莽草酸对大鼠腹腔肥大细胞脱颗粒及抗炎作用研究

目的:探讨资木瓜总提物中莽草酸(Shikimic acid,SA)对C48/80(Compound 48/80)刺激大鼠腹腔肥大细胞(Rat peritoneal mast cells,RPMC)脱颗粒的影响及抗炎作用。方法:HPLC 法进行资木瓜总提物中莽草酸成分的定性分析;采用甲苯胺蓝(Toluidine blue,TB)、免疫组化及电镜法进行RPMC 鉴定;CCK-8 法检测各浓度组RPMC 细胞的增殖情况;底物法检测β-己糖苷酶的释放率;ELISA 法检测细胞上清中组胺的含量。结果:SA 在0 ~90 μg/ ml 时,对RPMC 生长无显著影响;与阳性对照组比较,SA 能有效抑制β-己糖苷酶的释放和抑制组胺的分泌。结论:SA 通过抑制C48/80 刺激的RPMC 脱颗粒而抑制炎症介质组胺的释放,进而发挥抗炎作用。     

羧胺三唑对RBL-2H3细胞活化脱颗粒的影响

目的研究羧胺三唑(CAI)对RBL-2H3肥大细胞增殖、凋亡及活化脱颗粒的影响,探索CAI的抗感染作用机制。方法以C48/80诱导RBL-2H3细胞活化脱颗粒模型,中性红染色法观察细胞脱颗粒的形态学,分别用ELISA法和底物显色法检测细胞培养上清中组胺和β-氨基己糖苷酶的释放水平,CCK-8法测定细胞活力,Hoechst 33342荧光染色法检测细胞凋亡。结果与对照组相比,10、20和40μmol/L CAI能够不同程度抑制C48/80诱导的RBL-2H3细胞脱颗粒反应,20和40μmol/L CAI能够降低C48/80诱导的组胺释放(P0.01),40μmol/L CAI能够降低β-氨基己糖苷酶的释放(P0.01)。另外,所用各浓度的CAI对细胞增殖和凋亡均无明显影响。结论 CAI能有效抑制RBL-2H3肥大细胞的活化脱颗粒,此作用并不是通过细胞毒发挥作用的。CAI可能部分通过下调肥大细胞的功能活化,发挥其抗感染作用。     

羌活水提取物对肥大细胞活化的影响

目的:探讨羌活水提取物(EN)对肥大细胞活化的影响及其机制。方法:体外获取大鼠腹腔肥大细胞(RPMC),观察EN不同剂量对anti-DNP IgE诱导肥大细胞活化的影响,酶联法检测anti-DNP IgE介导的肥大细胞组胺释放,酶联免疫吸附测定法检测肥大细胞肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)释放,免疫印迹检测丝氨酸/苏氨酸蛋白激酶(Akt)、核转录因子κB(NF-κB)p65蛋白的表达。结果:体外anti-DNP IgE明显促进RPMC组胺,肿瘤坏死因子-α(TNF-α)和白介素6(IL-6)的释放,诱导Akt磷酸化和NF-κB p65活化,羌活水提取物高(200μg/L)、中(100μg/L)浓度明显抑制肥大细胞组胺,TNF-α和IL-6的释放,并抑制Akt磷酸化和NF-κB p65活化。结论:羌活水提取物通过调节Akt,NF-κB p65信号通路抑制肥大细胞介导的过敏性炎症。     

血管活性肠多肽及降钙素基因相关肽对大鼠腹腔肥大细胞组胺释放的作用

目的:研究血管活性肠多肽(Vasoactive intestinal peptide,VIP)及降钙素基因相关肽(Calcitonin gene relatedpeptide,CGRP)对大鼠腹腔肥大细胞脱颗粒的诱导作用;了解神经多肽与肥大细胞的相互关系。方法:分离、纯化SD大鼠腹腔肥大细胞;应用不同浓度的VIP和CGRP作用于大鼠腹腔肥大细胞后,同位素放射液态闪烁法检测肥大细胞的组胺释放、45Ca摄入的变化;同时观察大鼠腹腔肥大细胞经5×10-6mol/L VIP受体抑制剂L-8-K处理后,对VIP诱导脱颗粒作用的影响。结果:5×10-6mol/L的VIP作用后大鼠腹腔肥大细胞组胺释放及45Ca摄入明显增加,并且这种变化与VIP呈剂量效应关系;CGRP对大鼠腹腔肥大细胞组胺释放无诱导作用;L-8-K作用后,肥大细胞对VIP的诱导活化作用无改变。结论:VIP可引起肥大细胞钙内流增加,进一步诱导肥大细胞脱颗粒、释放组胺等生物活性物质,产生生物学效应;这种作用是受体非依赖性的,且与VIP的分子构型有关。     

组胺、类胰蛋白酶、β-己糖胺酶在肥大细胞体外释放过程中的相互关系

目的:探索组胺、类胰蛋白酶、β-己糖胺酶在肥大细胞体外释放过程中的相互关系,筛选反映肥大细胞体外释放的最优指标.方法:肥大细胞体外致敏、激发,分光光度法检测组胺、类胰蛋白酶、β-己糖胺酶3种细胞介质.结果:组胺、类胰蛋白酶、β-己糖胺酶在肥大细胞体外释放的过程中能够实现平行释放,但是组胺的释放不够稳定、重复性较差和另外两种细胞介质相比有统计学差异(P<0.05).类胰蛋白酶和β-己糖胺酶二者释放的相似度较高,稳定性较好,其中类胰蛋白酶在细胞上清中含量最高,检测更为容易.结论:以类胰蛋白酶、β-己糖胺酶来反映肥大细胞脱颗粒程度比组胺更精确、更稳定,类胰蛋白酶是最优指标.     

大鼠IgE依赖组胺释放因子基因的克隆表达及其功能研究

目的:获得原核表达的可溶性大鼠IgE依赖组胺释放因子(rHRF),并检测其诱发大鼠致敏肥大细胞释放组胺的功能.方法:克隆Wistar大鼠rHRF基因完整编码区序列并在BL21细胞中进行原核表达,纯化的可溶性重组rHRF刺激卵清蛋白变应原致敏的大鼠肺肥大细胞,利用荧光分光光度法测定组胺释放量.结果:测序证实克隆基因与GenBank中大鼠IgE依赖组胺释放因子(又称翻译控制肿瘤蛋白)mRNA序列的一致性为97%,推测的氨基酸序列一致性为95%.SDS-PAGE显示重组rHRF相对分子质量(Mr)约为24 000;重组rHRF诱发大鼠致敏肥大细胞释放组胺不依赖变应原,且呈剂量依赖关系. 结论:rHRF具有不依赖变应原诱发大鼠致敏肥大细胞释放组胺的功能,可能在Ⅰ型超敏反应过程中发挥重要作用,为进一步研究rHRF的免疫学功能打下基础.     

大鼠腹膜源肥大细胞外泌体的分离、培养与鉴定

目的：分离、培养及鉴定大鼠腹膜源肥大细胞外泌体。方法：提取8～10周龄SPF级雌性SD大鼠腹腔灌洗液,使用Percoll密度梯度分离法分离灌洗液中的肥大细胞,通过甲苯胺蓝染色鉴定肥大细胞,在细胞培养板中加入白细胞介素3和干细胞生长因子诱导肥大细胞成熟,收集细胞上清液,采用超速离心法分离上清液中的外泌体,通过透射电镜,纳米流式细胞仪从外泌体形态、粒径和表面标志蛋白三个方面鉴定外泌体。结果：分离得到的肥大细胞被甲苯胺蓝染成蓝紫色,分离的肥大细胞外泌体呈典型盘状,粒径分布范围为30～150 nm,平均粒径为75.33 nm,外泌体表面标志蛋白CD9和CD81呈阳性表达。结论：Percoll密度梯度分离法联合超速离心法分离大鼠腹腔灌洗液中肥大细胞外泌体的方法是可行的。     

内脏高敏感模型中肥大细胞活性的改变及5-羟色胺的作用

目的　证实内脏高敏感模型中肥大细胞活性增加。探讨 5 -羟色胺 (5 -HT)影响肥大细胞脱颗粒的依据。方法　用腹腔注射鸡卵清白蛋白制备内脏高敏感大鼠随机分为A组和B组 ,空白对照组随机分为C组和D组。提取内脏高敏感大鼠和对照组大鼠的腹腔肥大细胞 ,用抗原和抗血清孵育刺激肥大细胞脱颗粒。计数肥大细胞脱颗粒率 ,并用荧光法检测其组胺释放率。比较经不含 5 HT孵育液组 (A组和D组 )和富含 5 HT孵育液 (B组和C组 )孵育过夜的肥大细胞脱颗粒率和组胺释放率。结果　在抗原孵育后 ,致敏肥大细胞的脱颗粒率A组平均为 4 2± 14 (% ) ,B…     

银杏叶提取物对于BPA活化RBL-2H3分泌IL-13的影响

目的:探讨银杏叶提取物(Extract of Ginkgo biloba,以下简称EGB)对于BPA(Bisphenol A,双酚A)活化RBL-2H3(简称RBL)分泌白细胞介素13(IL-13)和蛋白激酶C(PKC)活化的影响.方法:利用BPA激活RBL,通过测定β-氨基己糖苷酶了解活化RBL脱颗粒情况,利用ELISA测定IL-13,Western blot检测RBL胞膜与胞浆PKC比值了解PKC活化情况.结果:经BPA活化后,RBL释放的β-氨基己糖苷酶上升到41.6％,IL-13分泌量上升到95.4 pg/ml,加入EGB(浓度为160μg/ml)后,RBL释放的β-氨基己糖苷酶降低到18.7％,分泌的IL-13降低到45.3 pg/ml,同时PKC活化也受到了明显的抑制.结论:EGB通过抑制RBL细胞的PKC活化,进而抑制活化RBL脱颗粒和IL-13分泌,提示EGB治疗哮喘的作用可能与其抑制肥大细胞PKC活化和分泌IL-13有关.     

倍增浓度的依巴斯汀有效抑制肥大细胞脱颗粒及细胞因子释放

目的研究依巴斯汀对肥大细胞活化剂C48/80体外诱导肥大细胞脱颗粒和细胞因子释放效应的影响,探讨依巴斯汀治疗荨麻疹的作用机制。方法依巴斯汀处理C48/80诱导活化的肥大细胞,通过MTT法检测细胞活力,β-氨基己糖苷酶脱颗粒试验检测细胞脱颗粒效应,ELISA法检测细胞分泌TNF-α、IL-4、IL-1β、LTB4、LTE4和组胺水平,q-PCR检测肥大细胞MCP-1、MCP-3、eotaxin、TNF-α、IL-4、IL-1β、IL-5及IL-6 m RNA表达水平。结果依巴斯汀浓度加倍处理肥大细胞能抑制其脱颗粒,其中4倍浓度(4×10^-4mmol/L)时抑制脱颗粒有极其显著差异(P<0.001)。依巴斯汀能抑制肥大细胞分泌IL-1β、TNF-α、IL-4、LTE4和组胺,但LTB4分泌没有差异;以浓度依赖方式显著抑制MCP-1、MCP-3、eotaxin、TNF-α、IL-4、IL-1β、IL-5及IL-6在m RNA表达(P<0.001),其中4倍和8倍剂量效应最显著,但二者抑制效果相当。结论依巴斯汀能显著抑制C48/80诱导肥大细胞脱颗粒、细胞因子和趋化因子的转录表达从而抑制炎症过程,但4倍浓度抑制效果最好,故研究结果为临床荨麻疹治疗提供了新思考。     

Differential staining of mast cells with toluidine blue

Abstract

Mast cells play a significant role in inflammatory diseases such as asthma, inflammatory bowel disease, and autoimmune diseases. Inhibition of c-kit receptor tyrosine kinase, a growth factor receptor, significantly reduces mast cell numbers. The purpose of this study was to determine the effect of Compound X (a c-kit inhibitor) on mast cell numbers in rats. Connective tissue mast cells (CTMCs) and mucosal mast cells (MMCs) have differing histochemical characteristics which presents a challenge when staining for quantification by semi-automated image analysis. CTMCs are present in tissues such as tongue and skin and will stain readily in tissues fixed routinely. In contrast, MMCs, such as those present in the intestinal mucosa, are sensitive to fixation. Brief fixation in Carnoy’s solution, although seldom used due to its composition (a mixture of ethanol, chloroform, and acetic acid), was employed to fix tissues for MMC staining, while tissues for CTMC demonstration were fixed in 10% neutral buffered formalin. An enzyme histochemistry method, napthol AS-D choloroacetate (specific esterase), was briefly considered for staining; however, granulocytes stained along with mast cells, requiring manual identification and exclusion, thereby rendering the method incompatible with automated means of quantification. Instead, staining was performed using two different toluidine blue methods which have proven conducive to semi-automated image analysis techniques. CTMCs were stained using Luna’s toluidine blue, while MMCs were stained with Matsson’s toluidine blue modification. In summary, the selected methods, based upon a conventional stain, were easy to do and successfully identified both populations of mast cells for quantification by image analysis.     

绵羊肥大细胞中类胰蛋白酶的证实

用甲苯胺蓝和阿尔辛蓝常规组织化学方法及特异性酶底物鉴定人肥大细胞类胰蛋白酶的酶组织化学技术，采用小鼠抗人肥大细胞类胰蛋白酶单克隆抗体，（ＡＡ１、ＡＡ３和ＡＡ５）通过间接免疫过的经物酶技术对绵羊肥大细胞的组织化学特性进行研究，酶组织化学技术及免疫组织化学技术均首次证实了绵羊肥大细胞颗粒中含有类胰蛋白酶，且该酶可作为绵羊肥大细胞的特异性标志，对于绵羊肥大细胞的常规组织化学染色，Ｃａｒｎｏｙ液及中性缓门     

Mast cell density and subtypes in the skin of dogs with atopic dermatitis

Skin biopsies from seven dogs with atopic dermatitis and 13 dogs with no clinical or histological evidence of skin diseases were examined. The study of the atopic dogs included 11 biopsy samples of nonlesional skin and 15 samples of lesional skin. One section of each tissue sample was stained with haematoxylin and eosin and another with toluidine blue to demonstrate the sulphated acid glycosaminoglycans in mast cell (MC) granules. To investigate MC subtypes, the MC-specific proteases tryptase and chymase were examined by a double enzyme-immunohistochemical staining technique. With the double labelling technique a significantly lower mast cell density was demonstrated in lesional (P = 0.0023) and nonlesional (P = 0.0004) skin samples of the atopic dogs than in the skin of control dogs. In the dermis of control dogs, a median mast cell density of 31.2 MC/mm2 was detected with the toluidine blue staining method and of 27.5 MC/mm2 with the double labelling technique. In lesional dermis of atopic dogs 29.8 MC/mm2 were seen with toluidine blue while only 12.4 MC/mm2 were stained with the double labelling method (P = 0.0027). A similar difference was observed in nonlesional dermis samples, in which a mast cell density of 23.3 MC/mm2 was detected with toluidine blue but only 6.4 MC/mm2 with the double labelling method (P = 0.0127). The data provide evidence that mast cell granule heterogeneity exists in the dog and suggests that degranulation occurs selectively, depending on the pathological condition of the canine skin. Further investigations on the pathophysiological role of mast cell subtypes may help to elucidate the pathogenesis of atopic dermatitis.     

Mast cells in the sheep, hedgehog and rat forebrain

The study was designed to reveal the distribution of various mast cell types in the forebrain of the adult sheep, hedgehog and rat. Based on their histochemical and immunocytochemical characteristics, mast cells were categorised as (1) connective tissue-type mast cells, staining metachromatically purple with the toluidine blue method, or pale red with the Alcian blue/safranin method, (2) mucosal-type or immature mast cells staining blue with the Alcian blue/safranin method and (3) serotonin immunopositive mast cells. All 3 types of brain mast cells in all species studied were located in both white and grey matter, often associated with intraparenchymal blood vessels. Their distribution pattern exhibited interspecies differences, while their number varied considerably not only between species but also between individuals of each species. A distributional left-right asymmetry, with more cells present on the left side, was observed in all species studied but it was most prominent in the sheep brain. In the sheep, mast cells were abundantly distributed in forebrain areas, while in the hedgehog and the rat forebrain, mast cells were less widely distributed and were relatively or substantially fewer in number respectively. A limited number of brain mast cells, in all 3 species, but primarily in the rat, were found to react both immunocytochemically to 5-HT antibody and histochemically with Alcian blue/safranin staining.     

大鼠胸腺实质中的肥大细胞—异染性,嗜银性,粘多糖和酶组织化学的研究

从异染，嗜银性，粘多糖和酶组织化学方面探讨了大鼠胸腺实质中的肥大细胞的异质性。结果表明；肥大细胞分布于皮，髓交界区或散在于皮质和髓质中。甲苯胺蓝染色和多糖组织化示；至少存在两种类型的肥大细胞，结缔组织在细胞和粘膜大细胞。在酶组织化学方面其也存在着异质性，尤其发现肥大细胞内含有ＡｃｈＥ阳性反应颗粒存在。此外，还发现肥大伴随ＡｃｈＥ阳性神经纤维分布，两者紧密接触，互相联接。     

The mast cells of the human uterus

Staining of mast cells in the human uterus has been studied using four fixatives and five staining methods to determine whether there are subpopulations of mucosal (endometrial) and connective tissue (myometrial) mast cells, and to discover how they can best be demonstrated. Following formalin fixation none of the staining methods showed maximum staining of mast cells in either endometrium or myometrium. The best demonstration of uterine mast cells is by fixation with either isotonic formol acetic acid or Mota's basic lead acetate followed by staining with the long toluidine blue technique. Although the degree of MC understaining following formalin fixation was greater for the endometrium than for the myometrium this is inadequate evidence to designate two cell populations. The findings suggest that the mast cells of the human uterus are all one population but show heterogeneity of histological properties possibly related to their functional state.     

Passive sensitization of human intestinal mast cells

Dispersed human intestinal mast cells were used for passive sensitization experiments. Eight biopsies (9.7±1.2 mg/biopsy) of human duodenum were collected from non-atopic children (5) and adults (5). The tissue was dispersed mechanically and enzymatically to yield single cell suspensions. The method produced 2×10³ mast cells per mg wet weight of tissue in a purity of 2.8%. Passive sensitization of the mast cells was performed with the patients' own plasma and plasma obtained from atopic donors. The non-atopic mast cells were able to bind the allergen-specific IgE. In addition, passive sensitization with atopic donor-plasma enhanced the cell sensitivity and cell reactivity to anti-IgE challenge, but had no effect on the cellular response to the ionophore A23187. The study shows that the enzymatic dispersion of human intestinal mast cells produces functionally intact mast cells with preserved Fc-receptors which can be passively sensitized.     

Phenotypic expression of mast cell granule proteinases. Distribution of mast cell proteinases I and II in the rat digestive system.

The distribution of mast cell granule proteinases in the rat digestive system was determined immunohistochemically. The population of toluidine blue-staining mast cells was accounted for by cells containing either rat mast cell proteinase I (RMCPI) or rat mast cell proteinase II (RMCPII). Granules in greater than 90% of RMCPI-containing cells stained red after the Alcian blue/safranin sequence, whereas all RMCPII-containing mast cells stained blue. The red/RMCPI phenotype was typical of the connective tissue mast cells (CTMC) that populate the proximal, non-mucosal, regions of the digestive system, and was also abundant in the serosa and in rectal and gastric muscularis. The blue/RMCPII phenotype, absent from non-mucosal sites except for rare cells in intestinal submucosa and muscularis, predominated in all mucosal tissues and resembled mucosal mast cells (MMC). A third, minor population of cells containing RMCPI but staining blue in the Alcian blue/safranin sequence was detected in both non-mucosal and mucosal tissues. Blue/RMCPI mast cells were rare in the small intestine but more frequent in the mucosa of the stomach and large intestine and in the connective tissues. It is suggested that granule proteinase phenotyping may provide an alternative technique in the analysis of mast cell heterogeneity.     

Mast cell heterogeneity in dog skin

The degree of metachromasia of mast cell granules is known to vary with the type of tissue fixation and among different tissues and species. The present study sought to determine whether mast cells in dog skin are heterogeneous with respect to fixation and staining properties. We performed skin biopsies in six anesthetized, atopic dogs and one mongrel dog. One biopsy was fixed in formalin and a second, from a parallel abdominal site, was fixed in basic lead acetate (Mota's solution). Adjacent sections from each biopsy were stained with alcian blue (1%, pH 0.5) or for chloroacetate esterase activity. In alcian blue-stained sections, one-third fewer mast cells were detected in skin fixed in formalin (1,836 +/- 454 mast cells/mm3, mean +/- SEM) than in skin fixed in basic lead acetate (2,684 +/- 527 mast cells/mm3) (P less than 0.05). The chloroacetate esterase reaction detected the larger number of mast cells regardless of the fixative used. We conclude that mast cell heterogeneity, as demonstrated by metachromatic staining following different types of tissue fixation, exists in dog skin. "Typical" mast cells stain with alcian blue regardless of fixation; however, "atypical" mast cells exhibit metachromasia only after fixation in basic lead acetate. Both the typical and atypical types of mast cells have chloroacetate esterase activity.     

Identification of mast cells in bronchoalveolar lavage fluid

The present study was carried out to compare the effectiveness of different fixation and staining methods in the identification of mast cells obtained by bronchoalveolar lavage from patients with interstitial lung diseases. Cell preparations were fixed with formaldehyde or methanol. Mast cells were identified by metachromatic staining with May Grünwald Giemsa, Toluidine blue or Gallamine blue Giemsa. After formaldehyde fixation only a few mast cells were identified, regardless of the stain method used. In contrast, a significantly higher number of mast cells were observed after methanol fixation. Using this fixative, Toluidine blue stain showed a higher number of mast cells than May Grünwald Giemsa. However, there was no difference between Toluidine and Gallamine blue Giemsa in the number of cells observed. The easy identification of mast cells after staining with toluidine, combined with its easy application, suggest that Toluidine blue stain after methanol fixation is the most useful method for determining the presence of mast cells in lavage fluid.