青藤碱对RBL-2H3肥大细胞增殖凋亡以及活化脱颗粒的影响

目的 通过研究青藤碱对肥大细胞增殖凋亡以及活化脱颗粒的影响,深入探讨青藤碱的抗炎作用机理.方法 应用细胞培养,流式细胞术等方法,观察青藤碱等药物对体外培养的RBL-2H3肥大细胞增殖凋亡以及活化脱颗粒的影响.结果 青藤碱对肥大细胞增殖有明显的抑制作用,其IG50约为1 mmol/L,并能促进肥大细胞凋亡,且对RBL-2H3肥大细胞活化脱颗粒有较强的抑制作用.结论青藤碱可能部分通过下调肥大细胞功能发挥抗炎抗风湿作用.     

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。     

RBL-2H3细胞诊断过敏性休克的可行性初探

目的 :探索诊断过敏性休克的体外检测方法。方法 :建立豚鼠过敏性休克模型 ,培养RBL 2H3细胞株 ,用致敏动物血清在体外诱导该细胞脱颗粒 ,观察其形态改变及测定组胺释放量与细胞表面AnnexinV标记阳性率。结果 :RBL 2H3细胞在致敏血清刺激下活化 ,在抗原再次攻击后可以脱颗粒 ,AnnexinV阳性率与组胺释放量呈正比 ,二者与致敏血清的滴度呈剂量依赖关系。结论 :RBL 2H3可望用于过敏性疾病诊断及变应原筛选 ,组胺测定及AnnexinV阳性率均为较好的测定指标     

The Distribution of Synaptotagmin Ⅱ in RBL-2H3 and Its Regulation on Exocytosis of Lysosomes in RBL-2H3

Synaptotagmin （Syt） constitutes a family of membrane-trafficking proteins, so far nearly 20 Syts have been discovered. Extensive work showed that synatotagmins were a potential Ca^2＋ sensor for regulated exocytosis. This study was to investigate the expression and location of synaptotagmin II （Syt2） in RBL-2H3 （RBL） and its role in regulating exocytosis of RBL. The expression of Syt2 in RBL was confirmed by Western blot. The recombinant expression vector pEGFP-N1-Syt2 was constructed and transfected into RBL by electroporation, the stable transfectant RBL-Syt2-S expressing fusion protein Syt2-EGFP were obtained and Syt2 was highly concentrated at plasma membrane with little detected in cytoplasm. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. Compared with control, the release of cathepsin D by RBL-Syt2-S was markedly decreased. The results indicated that Syt2 played a negative regulation in exocytosis of lysosomes in RBL. Cellular ＆ Molecular Immunology. 2005;2（3）：205-209.     

大鼠RBL-2H3细胞瞬时感受器电位M7通道参与细胞存活及凋亡**☆

背景：瞬时感受器电位M7是肥大细胞上重要的钙离子通道,但其在肥大细胞存活及凋亡过程中的作用仍未明确。 目的：观察瞬时感受器电位M7通道对大鼠RBL-2H3细胞存活及凋亡的影响。 方法：取对数生长期RBL-2H3细胞,①分别以50,100,200 μmol/L瞬时感受器电位通道阻断剂2-APB进行干预,并以0.1%DMSO干预的细胞及正常培养的细胞作对照。②以瞬时感受器电位M7-siRNA反转录病毒载体转染RBL-2H3细胞,并设立空载体组和正常对照组。 结果与结论：经过100,200 μmol/L的2-APB作用72 h后,RBL-2H3细胞数减少,吸光度值降低(P < 0.05),细胞凋亡增多,早期凋亡率和总凋亡率增加(P < 0.05);RBL-2H3细胞转染si-瞬时感受器电位M7 72 h 后,吸光度值降低(P < 0.05),细胞凋亡增多,早期凋亡率和总凋亡率增加(P < 0.05)。说明瞬时感受器电位M7通道参与了RBL-2H3细胞存活和凋亡过程。       

隐丹参酮对肾癌干细胞增殖和凋亡的影响

BACKGROUND: Previous studies have found that cryptotanshinone represses multiple tumors, but little is reported on its effect on renal carcinoma. OBJECTIVE: To explore the effect of cryptotanshinone on the proliferation and apoptosis of the renal carcinoma stem cells. METHODS: CD133+ renal carcinoma stem cells were separated from OS-RC-2 cells by immunomagnetic bead separation. Effects of 0, 0.2, 1, 5 mg/L cryptotanshinone on the proliferation and apoptosis of CD133+ renal carcinoma stem cells were detected by MTT and flow cytometry, respectively. Expression levels of Ki67, Bcl-2, Caspase-3 and p-Caspase-3 protein were detected by western blot assay. RESULTS AND CONCLUSION: After magnetic cell sorting, the percentage of CD133+ cells was increased from 6.32% to 82.73%, and there was a significant difference before and after cell sorting (P < 0.001). Cryptotanshinone could repress the proliferation of CD133+ renal carcinoma stem cells and promote cell apoptosis in a dose-dependent manner. The protein expression levels of Ki67 and Bcl-2 in the 5 mg/L cryptotanshinone group were significantly decreased compared with the control group, while the protein expression level of p-Caspase-3 protein was significantly increased. In addition, there was no difference in the protein expression of Caspase-3 between cryptotanshinone and control group. These findings indicate that cryptotanshinone may be a potent anticancer drug for the treatment of renal carcinoma by inhibiting expression of Ki67 and Bcl-2 and promoting protein expression of p-Caspase-3.  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程      

反转录病毒介导靶向瞬时感受器电位M7基因siRNA抑制RBL-2H3细胞的活化

背景：钙离子在肥大细胞活化后的脱颗粒反应起重要作用。瞬时感受器电位M7(transient receptor potential melastatin 7,TRPM7)是肥大细胞重要的候选通道。 目的：构建携带大鼠靶向TRPM7-siRNA的反转录病毒载体并检测其对大鼠RBL-2H3细胞抗原活化的影响。 方法：实验设计3个TRPM7-siRNA序列和1个无关对照序列,克隆到酶切的pSuper-retro-neo-GFP反转录病毒载体,用重组质粒pSuper-retro-neo-GFP-shTRPM7-(1, 2, 3) 采用脂质体Lipofectamine 2000转染RBL-2H3细胞,采用Western blot检测干扰效率。筛选出最有效的pSuper-retro-neo-GFP-siTRMP7与包装质粒共转染293FT细胞生成反转录病毒并感染RBL-2H3细胞,荧光实时定量PCR及Western blot检测TRPM7-siRNA的沉默效果。检测β-氨基已糖苷酶活性探讨RBL-2H3细胞抗原活化程度的改变。 结果与结论：转染后的各组细胞中siTRPM7-3转染组的沉默效率最高(P < 0.05)。pSuper-retro-neo-GFP-siTRMP7-3干扰组的TRPM7基因的mRNA水平和蛋白水平显著下调,致敏后其β-氨基已糖苷酶活性明显降低(P  < 0.05)。结果提示,降低TRPM7基因的表达可抑制RBL-2H3细胞的抗原活化。     

大鼠IgECH2-3-FasL融合蛋白基因转染对RBL-2H3的影响

目的：探索大鼠IgECH2-3-FasL融合蛋白对大鼠肥大细胞株RBL-2H3诱导凋亡作用和抑制其脱颗粒作用。方法：将本室构建的真核表达质粒peDNA3．1／IgECH2-3-FasL转染肥大细胞株RBL-2H3，RT-PCR及免疫印迹法鉴定大鼠IgECI-12-3-FasL融合蛋白的表达，ArmexinV流式细胞分析融合蛋白对RBL-2H3的凋亡作用，通过组胺释放率的测定，分析融合蛋白对RBL-2H3脱颗粒的阻断作用。结果：大鼠IgECI-12-3-FasL以融合蛋白的形式在RBL-2H3上获得表达，并且诱导RBL-2H3凋亡，部分阻断了RBL-2H3的脱颗粒。结论：大鼠IgECI-12-3-FasL融合蛋白具有促进肥大细胞凋亡和抑制肥大细胞脱颗粒的双重作用。     

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.     

银杏叶提取物对双酚A活化RBL-2H3细胞株表达IL-4和TNF-α的影响

探讨银杏叶提取物(extract of Ginkgo biloba,EGB)对于双酚A(Bisphenol A,BPA)活化RBL-2H3(简称:RBL)表达IL-4和TNF-α的影响。利用BPA激活RBL,通过测定β-氨基已糖苷酶和RT-PCR,研究EGB对于活化RBL脱颗粒以及表达IL-4和TNF-α的影响。结果显示,经BPA活化的RBL,加入EGB(浓度为160μg/ml)后,其β-氨基已糖苷酶释放仅为对照的45%,其IL-4mRNA表达量降低到对照的33.5%,TNF-αmRNA表达量降低到对照的26.8%。结果说明EGB极大地抑制了活化RBL的脱颗粒,同时对于活化RBL表达IL-4和TNF-α也具有极大的抑制作用,提示EGB治疗哮喘的作用与其抑制肥大细胞表达IL-4和TNF-α有关。     

The role of phosphatidylinositol 4-kinase type IIα in degranulation of RBL-2H3 cells

Objective and Design We have studied the role of phosphatidylinositol 4-kinase IIα (PI4KIIα) in activation of rat basophilic leukemia (RBL-2H3) cells. Materials and Methods Antigen-mediated intracellular Ca²⁺ concentration ([Ca²⁺]_i) increase and β-hexosaminidase secretion were measured using RBL-2H3 cells stably expressing PI4KIIα-yellow fluorescent protein (YFP) or its kinase-deficient mutant PI4KIIα (K151A)-YFP. Results Neither PI4KIIα-YFP nor PI4KIIα (K151A)-YFP were distributed on the plasma membranes but on the exocytotic vesicles. The RBL-2H3 cells stably expressing PI4KIIα-YFP showed significantly enhanced β-hexosaminidase secretion but not an increase in [Ca²⁺]_i after antigen stimulation. The cells with PI4KIIα (K151A)-YFP showed no change in the [Ca²⁺]_i increase nor degranulation. The promotion of secretion by PI4KIIα-YFP was not observed using co-stimulation with Ca²⁺ ionophore and the protein kinase C activator, phorbol myristate acetate. Conclusions These results suggest that PI4KIIα plays a role in the exocytotic process downstream of Ca²⁺ signaling in antigen-mediated mast cell activation. Received 29 September 2005; returned for revision 31 October 2005; accepted by A. Falus 23 May 2006     

The characterization of tyrosine phosphorylation of 78 and 92 kDa proteins in rat basophilic RBL-2H3 cells

Previously, we have demonstrated that tyrosine phosphorylation of 78 and 92 kDa proteins in rat basophilic leukemia cells (RBL-2H3) is involved in a signal transduction system for high-affinity IgE receptor (FcRI)-mediated histamine secretion. However, it is not clarified whether the tyrosine phosphorylation of 78 and 92 kDa proteins in RBL-2H3 cells is regulated by activation of protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3-kinase). In this study, therefore, the effect of depletion of PKC in RBL-2H3 cells, or the influence of PKC, PI3-kinase and tyrosine kinase inhibitors on histamine release from RBL-2H3 cells was examined. The elimination of PKC in RBL-2H3 cells induced significant suppression of histamine release, although the tyrosine phosphorylation of 78 and 92 kDa proteins was not inhibited. The inhibition of histamine release was also observed by the treatment with a PKC inhibitor such as H-7, calphostin C, a PI3-kinase inhibitor such as wortmannin or a tyrosine kinase inhibitor such as ST638, genistein, hervimycin A, although the tyrosine phosphorylation of both proteins was inhibited by only ST638. These results suggest that the 78 kDa protein in RBL-2H3 cells is not identical to the protein-tyrosine kinase PTK72 and the tyrosine phosphorylation of 78 and 92 kDa proteins in RBL-2H3 cells occurs upstream of PKC and PI3-kinase activation or is regulated independently of the PKC- and PI3-kinase-dependent signaling pathway.accepted by M. Katori     

Inhibition of the Antigen-Induced Activation of RBL-2H3 Cells by Gab2 siRNA

Gab2 plays an important role in FcεRI mediated signal events which lead to degranulation from mast cells. The present study was designed to investigate the effect of the synthetic Gab2 （scaffolding adapter Grb2-associated binder 2） siRNA on the antigen-induced activation of RBL-2H3 cells. A double stranded siRNA against Gab2- mRNA was synthesized and transfected into RBL-2H3 cells. After 6 h, cells were then sensitized with dinitrophenyl （DNP）-specific IgE overnight and challenged with dinitrophenyl-human serum albumin （DNP-HSA） to induce mast cell degranulation before supernatants were collected. Effects of Gab2 siRNA on antigen-induced release of β-hexosaminidase and histamine, cytokine production and regulation of the proteins in the pathway were measured by enzymatic assay, EIA, ELISA and Western blotting. Treatment with Gab2 siRNA significantly decreased Gab2 expression, inhibited the FcεRI-mediated mast cell release of β-hexosaminidase and histamine, reduced the production of IL-4 and TNF-α and inhibited the phosphorylation of Akt, PKC8 and p38 mitogen-activated protein kinase （MAPK）. Data showed that Gab2 siRNA could suppress the antigen-induced activation of RBL-2H3 cells and suggested a possible mechanism through inhibition of signaling molecules downstream of Gab2 in the 2＋ FcεRI-mediated Ca -independent pathway. Furthermore, potential usefulness of Gab2 knock-down as a method for inhibition of mast cell-mediated allergic reactions was demonstrated. Cellular ＆ Molecular Immunology. 2008; 5（6）：433-438.     

Antibodies against human CD63 activate transfected rat basophilic leukemia (RBL-2H3) cells

CD63 is a widely expressed glycoprotein member of the transmembrane 4 superfamily (TM4SF) that is present on activated platelets, monocytes and macrophages and many non-lymphoid cells. It has been proposed that CD63 and other members of the TM4SF couple to intracellular signal transduction pathways and may have a role in cellular adhesion, proliferation and activation. We have investigated the functions of human CD63 by expression in the rat basophilic leukemia cell line, RBL-2H3, which has previously been reported to respond to antibodies against the rat homolog of CD63. Using a panel of antibodies against human CD63 we have shown that high levels of granular secretion from transfected RBL cells can be stimulated by some, but not all, of the antibodies. The specificity of this response suggests that these activating antibodies may be mimicking a natural ligand for CD63. The secretory response to crosslinking of the high affinity IgE receptor and also that to non-receptor stimuli (phorbol ester and calcium ionophore) is inhibited by an antibody that appears to recognise both human and rat homologs of CD63. These results suggest that stimulus-secretion coupling can occur through human CD63 and that RBL cells transfected with this protein will constitute a valuable tool in elucidating its function.     

Human serum IgE-mediated mast cell degranulation shows poor correlation to allergen-specific IgE content

BACKGROUND: Although allergen-specific IgE content in serum can be determined immunochemically, little is known about the relationship between this parameter and the strength of the degranulation response upon allergen triggering. OBJECTIVES: Analyse the degranulation capacity of immunochemically defined purified and serum IgE after challenge with anti-IgE or allergen using a rat mast cell line (RBL) transfected with the alpha-chain of the human high-affinity IgE receptor (FcepsilonRI). METHODS: Purified IgE specific for 4-hydroxy-3nitrophenylacetyl, purified IgE of unknown specificity, and sera from allergic patients sensitive to Dermatophagoides pteronyssinus and Dactylis glomerata were assessed. Degranulation was measured by a beta-hexosaminidase release assay after anti-IgE or allergen-specific challenge. RESULTS: For purified monoclonal IgE a significant correlation (r = 0.97) was found between the proportion of bound allergen-specific IgE and the strength of the degranulation response. In contrast, no correlation (r = 0.27) was detected after sensitization with serum IgE. CONCLUSION: Our studies demonstrate that mast cell activation mediated through IgE from allergic patients is a result of complex relationships that are not only dependent on allergen-specific IgE content but also relate to the capacity to efficiently sensitize and trigger the signalling responses that lead to degranulation.     

FasL基因转染对大鼠肥大细胞凋亡的影响

目的：观察FasL基因转染对大鼠肥大细胞凋亡的影响。方法：RT-PCR法扩增大鼠FasL穿膜区和胞外区cDNA，成功构建pcDNA3．1／FasL真核表达质粒，瞬时转染RBL-2H3，RT-PCR、免疫印迹法鉴定FasL在RBL-2H3上的表达，Annexinv流式细胞检测pcDNA3．1／FasL转染RBL-2H3后细胞的凋亡情况。结果：获得FasL穿膜区和胞外区eDNA，构建pcDNA3．1／FasL真核表达质粒，瞬时转染RBL-2H3后在细胞膜和上清均有FasL的存在，瞬时转染RBL-2H3后48小时，细胞发生凋亡。结论：吧大细胞可以通过Fas-FasL途径凋亡，为FasL基因应用于过敏性疾病的治疗提供依据。     

Inhibition of mediator release in RBL-2H3 cells by some H1-antagonist derived anti-allergic drugs: Relation to lipophilicity and membrane effects

In a model for mucosal mast cells (RBL-2H3 cells) a set H₁-antagonist derived anti-allergic drugs containing a diphenylmethyl piperazinyl moiety was examined for their ability to inhibit release of the mediator-hexosaminidase. Cells were activated with antigen or the calcium ionophore A23187, whether or not in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Oxatomide, hydroxyzine and cetirizine inhibit the antigen induced-hexosaminidase release. The release triggered by A23187, whether or not in combination with TPA is hardly influenced by the compounds. A biphasic dependence of the inhibition of exocytosis in RBL cells on lipophilicity is observed with the optimum at log P is 5–6. The extremely lipophilic compounds meclozine and buclizine are not active in this model. pH dependence of the effect of the drugs shows that especially the uncharged species are active in inhibiting exocytosis. The investigated compounds show an effect on phase transitions in L--phosphatidylcholine dipalmitoyl liposomes as assayed with differential scanning calorimetry (DSC). For the less extremely lipophilic compounds the induced changes in the phospholipid membranes increased with lipophilicity. The relation between structural features of the drug and the interaction with phospholipids is discussed in view of the DSC results. We conclude that location of the active drugs at the membrane or the membrane/protein interface is important for the inhibiting activity on exocytosis. This could affect several membrane related processes, which are abundant in the early phases of the IgE-mediated signal transduction process.Abbreviations RBL 2H3 cells, a subline of rat basophilic leukemia cells - TPA 12-O-tetradecanoylphorbol-13-acetate - A23187 calcium ionophore - LDH lactate dehydrogenase - DSC differential scanning calorimetry - DPPC L--phosphatidylcholine dipalmitoyl