依巴斯汀联合转移因子治疗慢性荨麻疹疗效观察

目的观察依巴斯汀联合转移因子治疗慢性荨麻疹的疗效。方法将120例慢性荨麻疹患者随机分为两组,治疗组63例,对照组57例。两纽均口服依巴斯汀10mg,1次／d,治疗组同时服用转移因子6mg,2次／d,疗程4周。结果治疗结束后,治疗组有效率为69．84％,对照组有效率为47．57％,两组疗效差异有统计学意义。结论依巴斯汀联合联合转移因子治疗慢性荨麻疹的疗效优于单用依巴斯汀治疗。     

苍耳子提取物抑制大鼠肥大细胞活化的机制研究

目的苍耳子具有抗过敏作用,但其作用机制尚未明确。本研究目的是利用能使肥大细胞活化的物质Compound 48/80,探讨苍耳子抗过敏作用机制。方法通过Compound 48/80诱导大鼠被动皮肤过敏反应(PCA)实验,用比色法测定苍耳子提取物在体内对肥大细胞影响。体外实验观察苍耳子提取物对Compound 48/80诱导组胺释放、细胞内钙摄入及细胞内cAMP水平的影响。结果Compound 48/80处理组对比正常组显著增强伊文思蓝的生成、组胺的释放、细胞内钙摄入以及减少cAMP量。苍耳子提取物前处理则明显抑制了Compound 48/80诱导的大鼠PCA实验,组胺的释放、细胞内钙摄入以及cAMP水平减少。结论苍耳子抗过敏作用可能与抑2+制肥大细胞内Ca摄入及增加cAMP量有关。     

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。     

Signaling pathway in nerve growth factor induced histamine release from rat mast cells

Objective and Design: We investigated a signal transduction pathway involved in NGF induced histamine secretion from mast cells. We compared this mechanism with the exocytosis induced by basic secretagogue compound 48/80.Materials and Methods: Isolated rat peritoneal mast cells were obtained from male Wistar rats. Histamine release was assayed spectrofl uorometrically.Results: We found that tyrosine kinase inhibitor genistein, phospholipase C (PLC) inhibitor U-73122, phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor wortmannin, protein kinase C (PKC) inhibitors, staurosporine and GF109203X, but not MAP kinase inhibitors, SB203580 and PD98059, reduce histamine secretion in NGF provoked mast cell degranulation. In compound 48/80 mediated degranulation, we confi rmed only the involvement of tyrosine kinase and PLC, but not PI-3 kinase, PKC and MAP kinases.Conclusions: Our results indicate that release of histamine from mast cells after stimulation with NGF is regulated by tyrosine kinase, PLC, PI-3 kinase and PKC, but not by MAP kinases. This biochemical pathway differs from that provoked by compound 48/80.Received 16 February 2005; accepted by A. Falus 12 May 2005     

Spontaneous histamine secretion during isolation of rat cardiac mast cells

Objektive: To test the hypothesis that spontaneous release of histamine occurring during an isolation protocol may modify responses of rat cardiac mast cells (connective tissue-type mast cells) to secretagogues.Methods: We assessed two protocols for enzymatic dispersion utilizing collagenase, hyaluronidase, and deoxyribonuclease; with protease (Protocol 1, n = 8) or without protease (Protocol 2, n = 3). Spontaneous release of histamine was quantified following mechanical and enzymatic dispersion of the whole heart.Results: Total histamine loss (Mean ± SEM) was 963±92 and 833±60 ng/g of tissue weight following Protocols 1 and 2. Percentages of histamine release from cell isolates following Protocol 1 were 40±5%, 41±6%, and 51±7% at 0, 30, and 300 ug/mL of compound 48/80.Conclusions: Enzymatic dispersion of cardiac mast cells affects their response to secretagogues.Received 8 January 2004; returned for revision 5 February 2004; accepted by A. Falus 22 March 2004     

Challenge of mast cells with increasing amounts of antigen induces desensitization

Background In vivo rush desensitization is a procedure widely employed to quickly desensitizo allergic patients by administering increasing doses of the offending antigen at short intervals. The mechanism(s) underlying this process and the possible role of mast cells in it have not been well delineated. Objective To define an ex vivo model for rush desensitization utilizing the mast cell-fibroblast coculture system. Methods Rat peritoneal mast cells were cultured on 3T3 fibroblasls and were pre-incubated with saturating or suboptimal concentrations of IgE anti-DNP antibodies. Mast cells were then repeatedly challenged at 30 min intervals, with increasing amounts (0.001 100ng/mL) of the antigen DNP-HSA, in the presence of calcium ions. Parallel control cultures were stimulated only once by each antigen concentration. In another set of experiments, mast cells were repeatedly activated with increasing concentrations (0.1-1000 ng/mL) of compound 48/80. Supernatants and cell sonicates were assessed for histamine content and the percentage of histamine released was calculated. Results When saturating concentrations of IgE anti-DNP antibodies were used, mast cells challenged repeatedly with DNP-HSA did not release significant amounts of histamine up to an antigen concentration of I ng/mL. At this stage they were partially desensitized, releasing only 108.3 . 17.1 ng histamine/plate (7.9±0.8%). A marked desensitization was observed at optimal antigen concentration (100 ng/mL). where experimental mast cells released only 45.6±10.9ng/plate, compared with 661.9±48.2ng/plate in firstly challenged cultures. Desensitization was probably not due to mast cells histamine depletion, since the cells still contained large amounts of histamine (579.5±108.6ng/plate) at the end of the procedure. A similar pattern of desensitization was observed when mast cell were preincubated with a subotimal concentration of IgE antibodies. Activation of mast cells with increasing amounts of the IgE-independent secretagogue, compound 48/80. did not cause desensitization since at each concentration both repetitively challenged and control cultures released similar amounts of histamine. Furthermore, challenge of antigen-desensitized mast cells with compound 48/80 caused the release of 75.9±4.9% histamine comparable to the percentage of histamine released from controls (79.5±6.7). Conclusion Repeated exposure of mast cells to gradually increasing amounts of antigen induces their refractoriness. This observation would suggest a role for mast cells in rush desensitization procedure in vivo. Our coculture system may serve as a useful model for studying this process.     

Freeze-fracture immunocytochemistry for intracellular localization of serotonin in mast cells stimulated with compound 48/80

Changes of intracellular localization of serotonin in rat mast cells were examined by freeze-fracture immunocytochemistry, to prevent the translocation of the serotonin antigen. Rat peritoneal cells including mast cells were stimulated in vitro with compound 48/80, at 17° C for 0, 30 or 60 s for exocytosis to occur. The mast cells were fixed, quickly frozen and freeze-fractured to expose the antigen on the fractured surface. They were immunostained with serotonin antibody, and the immunoreactions on the fractured surface were examined on ultrathin sections by electron microscopy. Unstimulated mast cells exhibited serotonin localization mostly in each intragranular matrix. In contrast, mast cells stimulated for 30 s exhibited increased serotonin in their intergranular cytoplasm. Mast cells showed more distinct immunoreactions in the cytoplasm where degranulation would be promoted after 60 s. It is suggested that intracellular release of serotonin occurred in the stimulated mast cells.     

Ultrastructural study of mast cells stimulated with compound 48/80 as revealed by quick-freezing method

The ultrastructure of mast cells stimulated with compound 48/80 was examined by quick-freezing and deep-etching (QF-DE) or freeze-substitution (QF-FS) methods. Peritoneal cells including mast cells of adult male rats were stimulated in vitro with compound 48/80 at 17° C for 0, 10, 30, 60 or 180 s. The QF-DE replicas revealed that the mast cells stimulated with compound 48/80 for 30 s decreased filamentous actin around secretory granules. In the QF-FS specimens, perigranular membranes in mast cells stimulated for 60 s formed pentalaminar structures between adjacent granules in their cytoplasm prior to degranulation. These findings suggest that preparatory states for degranulation occur in the whole cytoplasm of stimulated mast cells at early stages. Moreover, both QF-FS specimens and QF-DE replicas revealed a compact morphological appearance of discharged granules in the extra-cellular space, indicating the existence of considerable content within the granules. Skeletal structures in the granules were also demonstrated on QF-DE replicas prepared after extracting soluble elements from the cytoplasm. It is suggested that the granular contents associated with the skeletal structures are gradually detached from the discharged granules to ensure local concentration in the tissues.     

X-ray microanalysis of rat mast cells stimulated with compound 48/80 in combination with quick-freezing method

X-ray microanalysis was performed on rat mast cells prepared by quick-freezing, cryosectioning and freeze-drying (QF-FD) method, or quick-freezing and freeze-substitution (QF-FS) method. Peritoneal cells including mast cells were stimulated with compound 48/80 for 0, 10 or 30 s at 17° C, and the mast cells stimulated for 30 s started exocytosis. In X-ray spectra of the QF-FD specimen, mast cells stimulated for 10 s increased their levels of phosphorus, sodium and chlorine in the intergranular cytoplasm prior to exocytosis, and kept this increase until 30 s after stimulation. In the QF-FS specimen, where soluble elements were removed, peaks of phosphorus, sulphur and potassium could be detected as elements in X-ray spectra. Phosphorus increased and potassium decreased in intergranular cytoplasm of mast cells stimulated for 10 s, and these changes became more obvious after 30 s. However, supplemental increase of other cations such as sodium could not be detected in the QF-FS specimens.     

肥大细胞体外脱颗粒的检测方法

目的和方法：模拟体内的脱颗粒反应，分别通过荧光分光光度计和酶活力测定方法定量检测组胺和类胰蛋白酶释放，探讨类胰蛋白酶活力测定法检测肥大细胞脱颗粒的可行性。结果：致敏血清和肥大细胞共孵育后在抗原刺激下，引起组胺和类胰蛋白酶剂量依赖性释放，且二者释放是平行的。结论：与组胺的测定比较，类胰蛋白酶活力测定可以用来标记肥大细胞脱颗粒的程度，该法灵敏可靠，简单可行，为临床上诊断变态反应疾病和筛选变应原提供新的检测方法。