RBL-2H3细胞脱颗粒模型研究

RBL-2H3细胞是体外研究变态反应的常用细胞,为了建立RBL-2H3细胞脱颗粒检测模型,以C48/80作为阳性药物,分析比较了细胞形态观测法、台盼蓝染色法、β-氨基己糖苷酶释放率及钙离子质量浓度检测等细胞脱颗粒检测方法.通过对C48/80作用质量浓度及时间的摸索,建立C48/80刺激RBL-2H3细胞脱颗粒的最佳药物作用条件.结果表明,β-氨基己糖苷酶释放率与细胞脱颗粒程度存在显著量-效关系,且操作简单、结果稳定.C48/80工作质量浓度为60μg/mL,最佳作用时间40 min.在此基础上,优化了DNPIgE/DNP-BSA诱导细胞脱颗粒体系,0.4μg/mL DNP-IgE致敏RBL-2H3细胞过夜,10μg/mL DNP-BSA诱导细胞,细胞脱颗粒程度最严重.同时发现,不同释放介质对DNP-BSA诱导细胞脱颗粒有显著影响.该结果可为变态反应相关研究提供实验基础.     

抗过敏中草药活性成分的药效评价

肥大细胞受到抗原刺激后释放组胺是过敏反应的重要过程,对组胺释放的抑制能力是评价抗过敏药物的重要因素.选取多种传统中医记载具有抗过敏作用的中草药,检测了其在体外对肥大细胞脱颗粒并释放组胺的抑制效果.在具有明显效果的中草药中,土茯苓与芍药对于抑制肥大细胞脱颗粒最为突出, IC50分别为62.5μg/mL和113.8μg/mL.土茯苓的突出药效(药物浓度为500μg/mL时抑制率达到95.63;)未曾被报道,具有研究与开发价值.对其机理的初步研究表明蛋白激酶A可能是土茯苓抑制肥大细胞脱颗粒的重要靶点.     

维生素K1注射液安全性评价实验的探讨

本文旨在研究药物的过敏物质检测方法,为药品的安全性评价提供参考。通过对白色豚鼠(Cavia porcellus)主动全身过敏实验、被动皮肤过敏实验、RBL-2H3细胞脱颗粒实验、β-氨基己糖苷酶释放率实验和L929细胞毒性实验考察不同厂家维生素K1注射液的产品质量。结果发现,供试的10个厂家的产品质量存在明显差异。研究认为细胞实验作为一种快捷有效的过敏物质检测手段,可用于药品的质量评价。     

维生素K1注射液安全性评价实验的探讨

本文旨在研究药物的过敏物质检测方法,为药品的安全性评价提供参考。通过对白色豚鼠(Cavia porcellus)主动全身过敏实验、被动皮肤过敏实验、RBL-2H3细胞脱颗粒实验、β-氨基己糖苷酶释放率实验和L929细胞毒性实验考察不同厂家维生素K1注射液的产品质量。结果发现,供试的10个厂家的产品质量存在明显差异。研究认为细胞实验作为一种快捷有效的过敏物质检测手段,可用于药品的质量评价。
     

姜黄素体外抗流感病毒H1N1、H3N2实验研究

目的：观察姜黄素提取物体外对流感病毒H1N1、H3N2的抑制作用。方法：用狗肾细胞（MDCK）观察姜黄素体外对A型流感病毒H1N1亚型、A型流感病毒H3N2亚型病毒的直接杀灭作用。结果：姜黄素最大无毒浓度为12．5g／L,对H1N1有效抑制浓度为6．25g／L,对H3N2有效抑制浓度为1．56g／L。结论：姜黄素提取物确有明显的抗H1N1、H3N2复制作用。     

苹果原花青素对成骨细胞氧化损伤的保护作用

观察苹果原花青素对过氧化氢(H2O2)引起的成骨样细胞MC3T3-E1氧化损伤的保护作用。培养成骨样细胞MC3T3-E1,加入不同浓度的原花青素(20,30,40,50μg/mL),及H2O2(100,200,300,400μmol/L),采用MTT比色试验法观察MC3T3-E1的增殖情况;以40μg/mL的原花青素预处理细胞30 min,后加入300μmol/L H2O24 h,采用EdU试剂盒,碱性磷酸酶(ALP)活性检测法,活性氧(ROS)检测法,超氧化物歧化酶(SOD)活性检测法以及肿瘤坏死因子α(TNF-α)的ELISA检测法,观察原花青素对H2O2引起的成骨样细胞MC3T3-E1氧化损伤的保护作用。H2O2作用后,MC3T3-E1细胞的增殖受到抑制,其所产生的碱性磷酸酶(ALP)活性、SOD活性降低,而ROS与TNF-α浓度明显升高,但预先30 min给予细胞原花青素,就可以有效逆转这一趋势,苹果原花青素可有效减轻过H2O2引起的成骨样细胞MC3T3-E1氧化损伤。苹果原花青素有潜在的治疗骨质疏松症作用。     

美洲大蠊提取物CⅡ-3抗炎作用及机制研究

目的:研究美洲大蠊提取物CⅡ-3抗急性炎症的作用,并探讨其可能的作用机制。方法:采用二甲苯致大鼠耳肿胀和角叉菜胶致小鼠足跖肿胀法急性炎症模型考察美洲大蠊提取物CⅡ-3抗急性炎症的作用。结果:50、100mg/kg的CⅡ-3对大鼠耳肿胀有明显抑制作用;100、200mg/kg的CⅡ-3对小鼠足跖肿胀有非常明显的抑制作用,且能显著降低小鼠炎足浸泡液中炎症介质PGE2、组胺和MDA的含量,并能提高炎症组织中SOD的活性。结论:美洲大蠊提取物CⅡ-3对急性炎症具有较好的抑制作用,其抗炎作用可能与减少炎症介质的含量和清除自由基有关。     

细胞凋亡与NF-κB激活的信号传导研究

细胞凋亡是细胞受基因调控的主动死亡方式,对于生物体的正常发育及生理功能的维持具有重要意义。NF-κB是一类在动物细胞中广泛表达的转录因子,其主要生理功能之一是通过诱导凋亡抑制基因的转录,拮抗细胞凋亡的发生。NF-κB的活化调控是细胞生死抉择过程的关键机制之一。现已证明细胞凋亡及NF-κB活化的异常会导致多种人类疾病。本项目从凋亡的执行机制和调控机制两方面入手,对细胞凋亡的机理进行了研究。建立了基于爪蟾卵细胞提取物的非细胞凋亡诱导体系,并利用这一体系,发现爪蟾细胞凋亡特异核酸酶XAD及其特异性的抑制因子IXAD;首次证明磷酸肌酸和核质素在凋亡过程中的作用。克隆了3个新的凋亡诱导基因。在细胞凋亡的调节机制方面,克隆了7个分别作用于NF-κB活化信号通路不同步骤的新的调节基因,发现5个已知蛋白调节不同NF-κB活化途径的新功能。此外,在国际上较早开展了植物细胞凋亡的研究,首次报道体外培养植物细胞凋亡过程中有细胞色素C的释放。     

改性硫磺颗粒的制备及其改性沥青路用性能

通过在硫磺中添加H2S抑制剂来制备改性硫磺颗粒(MSP),采用亚甲基蓝分光光度法测定改性硫磺颗粒与沥青混合加热过程中H2S释放量,并讨论H2S主抑制剂、辅助抑制剂的种类和用量对H,s释放量的影响,优化改性硫磺颗粒配方.结果表明:二硫化物抑制剂是最佳的主抑制剂,且随主抑制剂用量的增加,H2S抑制效果增强;少量的协同抑制剂极大地提高了改性硫磺颗粒对H,s的抑制能力.在最佳配方下,160 ℃时改性硫磺颗粒与沥青共混时H2s释放量为0.03μg/g,与纯硫磺相比H2S释放量降至0.68%;150℃时不同配方改性硫磺颗粒与沥青共混时H,S释放量为0.012～0.156μg/g,而纯硫磺与沥青共混时H2S释放量为0.956μg/g.添加适量改性硫磺颗粒,沥青混合料的动稳定度由l 073次/mm提高至2410次/mm,冻融劈裂强度比由75.3%提高至84.4%.改性硫磺颗粒具有显著降低H2s释放量、增强路面强度、改善抗水损害能力等优点.     

石榴皮提取物体外抗氧化作用的研究

为研究石榴皮提取物的体外抗氧化作用。将石榴皮提取物分别配制成0.1、0.5、1、2、4 mg/m L不同浓度的溶液,用DPPH法进行体外抗氧化活性检测;用比色法测定抑制红细胞自氧化溶血,H2O2所致红细胞氧化溶血的作用。结果显示石榴皮提取物各剂量组均对DPPH自由基有抑制作用,且随剂量的增大抑制作用从52.4%到70.6%逐渐增强;各剂量组均能明显抑制红细胞自氧化溶血和H2O2诱导所致红细胞溶血,从0.5 mg/m L开始随剂量增大效应逐渐增强,有显著性差异。由此可知,石榴皮提取物具有良好的体外抗氧化作用,并对红细胞膜具有保护作用。     

Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases.

The high-affinity IgE receptor (Fc epsilon RI), which is expressed on the surface of mast cells and basophils, has a central role in immediate allergic responses. In the rat basophilic leukaemia cell line RBL-2H3, which is a model system for the analysis of Fc epsilon RI-mediated signal transduction, surface engagement of Fc epsilon RI induces histamine release and the tyrosine phosphorylation of several distinct proteins. Although the alpha, beta, and gamma subunits of Fc epsilon RI lack intrinsic tyrosine protein kinase (TPK) activity, a kinase that copurifies with Fc epsilon RI phosphorylates the beta and gamma subunits of the receptor on tyrosine residues. We report here that in RBL-2H3 cells, p56lyn and pp60c-src are activated after Fc epsilon RI crosslinking, and p56lyn coimmunoprecipitates with Fc epsilon RI. In the mouse mast-cell line PT-18, another cell type used to study FC epsilon RI-mediated signalling, tyrosine phosphorylation of proteins is also an immediate consequence of receptor crosslinking. Notably, the only detectable src protein-related TPK in PT-18 cells is p62c-yes, and it is this TPK that is activated on Fc epsilon RI engagement and coimmunoprecipitates with the receptor. Therefore, it seems that different src protein-related TPKs can associate with the same receptor and become activated after receptor engagement.     

Excitatory effect of histamine on neuronal activity of rat cerebellar fastigial nucleus in vitro

The cerebellar fastigial nucleus (FN) holds an important role in motor control and body balance. Previous studies have revealed that the nucleus is innervated by direct hypothalamocerebellar histaminergic fibers. However, the functional role of histaminergic projection in cerebellar FN has never been established. In this study, we investigated the effect of histamine on neuronal firing of cerebellar FN by using slice preparations. Sixty-five FN cells were recorded from 47 cerebellar slices, and a vast majority of the cells responded to histamine stimulation with an excitatory response (58/65, 89.2%). Perfusing slices with low-Ca2 /high-Mg2 medium did not block the histamine-induced excitation (n=10), supporting a direct postsynaptic action of histamine on the cells. Furthermore, the excitatory effect of histamine on FN neurons was not blocked by selective histamine H1 receptor antagonist triprolidine (n=15) or chlorpheniramine (n=10), but was effectively suppressed by ranitidine (n=15), a highly selective histamine H2 receptor antagonist. On the other hand, highly selective histamine H2 receptor agonist dimaprit (n=20) instead of histamine H1 receptor agonist 2-pyridylethylamine (n=16) mimicked the ex- citatory effect of histamine on FN neurons. The dimaprit-induced FN neuronal excitation was effectively antagonized by selective histamine H2 receptor antagonist ranitidine (n=13) but not influenced by se- lective histamine H1 receptor antagonist triprolidine (n=15). These results demonstrate that histamine excites cerebellar FN cells via the histamine H2 receptor mechanism and suggest that the hypotha- lamocerebellar histaminergic fibers may modulate cerebellar FN-mediated sensorimotor integration through their excitatory innervations on FN neurons.     

A hapten-specific chimaeric IgE antibody with human physiological effector function

Immunoglobulin E (IgE) has a central role in allergic reactions although it rarely exceeds 5 micrograms ml-1 even in the serum of severely allergic individuals. Both mast cells and basophils possess receptors which bind the Fc portion of IgE with high affinity; crosslinking of membrane-bound IgE by allergen results in degranulation of the cell and release of a variety of pharmacologically active mediator including histamine. Myeloma IgE has been successfully used to block the skin sensitizing activity of allergic sera; however, human myeloma IgE is clearly in limited supply. The emergence of techniques allowing the stable introduction of immunoglobulin gene DNA into myeloma cells has allowed us to construct a mouse cell line that secretes a chimaeric IgE, lambda 1 antibody whose heavy chain is composed of a human C epsilon constant region fused to a mouse variable (VH) region. This chimaeric IgE is specific for the hapten 4-hydroxy-3-nitro-phenacetyl (NP) and can, when crosslinked by antigen, trigger the degranulation of human basophils. When not crosslinked, however, the chimaeric IgE can prevent the passive sensitization of these cells by sera from allergic subjects.     

Potent ulcerogenic actions of platelet-activating factor on the stomach

Platelet-activating factor (PAF) is an endogenous phospholipid which has been implicated as a mediator of allergic and inflammatory processes. It is synthesized and released by neutrophils, platelets, macrophages, monocytes, basophils and endothelial cells, and is a potent platelet-aggregating agent, a vasodilator, increases vascular permeability, stimulates neutrophil aggregation and degranulation and induces release of lysosomal enzymes. A role for PAF in the hypotension associated with endotoxin shock and in necrotizing enterocolitis has recently been suggested. As there is an association between septic shock and acute gastric damage, we propose that PAF is an endogenous mediator of ulceration in the stomach. Indeed, as reported here, intravenous (i.v.) infusion of PAF to rats, at doses of 20-200 pmol per kg per min, resulted in the formation of extensive haemorrhagic erosions in the gastric mucosa. The ulcerogenic actions of PAF are not attributable solely to its hypotensive actions and were not mediated via effects on platelets or cyclooxygenase products, nor via histamine H1, H2 or alpha-adrenergic receptors. PAF is the most potent gastric ulcerogen yet described and its endogenous release may underlie or contribute to certain forms of gastric ulceration.     

Protein kinase C regulation of the receptor-coupled calcium signal in histamine-secreting rat basophilic leukaemia cells

It has been proposed that protein kinase C mediates cellular responses evoked by external stimuli, leading to alterations in internal free calcium concentrations. We have shown previously that histamine-secreting rat basophilic leukaemia cells (RBL-2H3), which degranulate on aggregation of the receptors for immunoglobulin IgE, contain a Ca2+- and phospholipid-dependent protein kinase (kinase C). The partially purified enzyme is activated directly by the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In intact RBL cells, TPA potentiates histamine release induced by the Ca2+-ionophore A23187 (similar to the synergy reported for platelets, neutrophils and rat peritoneal mast cells). Although TPA at concentrations below 15 nM synergizes with the antigen, higher TPA concentrations inhibit secretion. This selective inhibition suggested that kinase C is involved in both the activation and termination of the secretory process. To examine this possibility, we have determined the effect of TPA on changes in free cytosolic Ca2+ concentration during antigen-induced release. We report here that TPA completely blocks the increase in Ca2+ concentration induced by antigen. Our results strongly suggest that protein kinase C is involved in the regulation of receptor-dependent Ca2+ signalling.     

A novel class (H3) of histamine receptors on perivascular nerve terminals

Two types of histamine receptor, the H1- and H2-receptors, are found not only on vascular smooth muscle cells but on the perivascular autonomic nerve terminals. Activation of the prejunctional histamine receptors modifies transmitter release from the nerve terminals. Recently, histamine was shown to inhibit its own release from depolarized slices of rat cerebral cortex. This phenomenon was found to be mediated by a novel class of histamine receptor, the H3-receptor, that was pharmacologically distinct from the H1- and H2-receptors. Up to now, there has been no indication whether this third class of histamine receptor is present in any tissue other than the brain. We report here that histamine depresses sympathetic neurotransmission in the guinea-pig mesenteric artery by interacting with histamine H3-receptors on the perivascular nerve terminals. The pharmacological properties of these receptors are similar to those reported for the H3-receptors in the brain. Our data provide evidence for the existence of H3-receptors in the autonomic nervous system.     

高效液相色谱法测定贝壳类海洋生物中的多胺含量

利用柱前衍生高效液相色谱法测定了贝壳类海洋生物（蛤蜊、牡蛎、扇贝）中多胺（主要是指亚精胺（SPD）和精胺（SPM））的含量.采取多种提取试剂（如不同浓度的HC1和H2SO4、甲醇、水提醇沉等）考察了样品中多胺的提取效果,另外,确定了最佳提取时间和温度.结果表明：样品中多胺的最佳提取条件为6 mol·L-1HC1溶液,蛤蜊和牡蛎60℃下提取3h,扇贝提取时间4h为好.提取液用丹磺酰氯衍生,甲苯萃取后高效液相色谱分析.色谱条件：流动相为甲醇和水,采用梯度洗脱,柱温40℃,荧光检测.测定结果：亚精胺（SPD）的回归方程为Y=0.172 9＋0.2616 X（R=0.998 8）,检出限为0.48 pmol;精胺（SPM）的回归方程Y=0.074 0＋0.170 4X（R=0.999 2）,检出限为0.54 pmol.     

阳极液提取Mn~(2+)和NH_3-N的影响因素及其分析

以阳极液作为提取剂,从电解锰渣中提取Mn2+和NH3-N,考察了液固比、反应温度和反应时间等3个因素对Mn2+和NH3-N提取效果的影响.实验结果表明,阳极液对Mn2+的提取效果明显优于对NH3-N的提取效果.Mn2+的最佳提取条件是液固比(mL/g)为10∶1,在50℃下反应30min,提取率达72.1%;NH3-N的最佳提取条件是液固比为10∶1,在50℃下反应40min,提取率达45.6%.动力学研究表明,阳极液对电解锰渣中Mn2+的提取反应符合拟一级动力学方程,而对NH3-N的提取反应符合拟二级动力学方程.