咪唑斯汀对小鼠肥大细胞血管内皮细胞生长因子的调节作用

目的:探讨咪唑斯汀(mizolastine,MIZ)对小鼠肥大细胞(MC)血管内皮细胞生长因子(VEGF)的调节作用。方法:分离小鼠MC及IgE的获得、ELISA测定、RT-PCR。结果:(1)卵白蛋白诱导MC对VEGF的释放呈现时间依赖性增长。30min的抗原诱导,MC即可结构性表达VEGF,1h前MC对这些细胞因子的释放缓慢增长,1～4h时间段对VEGF的释放呈现急剧增长现象,峰值在4h,4h后VEGF释放值呈现滞长或逐渐降低,6h后比4h略降。(2)MIZ对IgE依赖方式诱导的小鼠MC的VEGF释放呈现浓度和时间依赖性抑制作用。(3)MC结构性表达VEGFmRNA,经抗原诱导后mRNA表达增强明显,经不同浓度MIZ阻断后表达明显降低,呈现浓度依赖的方式。结论:MIZ对体外MC展示很强的抑制其VEGF的表达能力,其作用机制可能存在多种途径。     

血管内皮生长因子及其受体在急性髓系白血病动物模型中表达的动态分析

本研究旨在探讨血管内皮生长因子（VEGF）及其受体（VEGFR）在急性髓系白血病（AML）发生和发展中的作用。建立急性髓系白血病（AML）SCID小鼠模型,采用酶联免疫吸附试验（ELISA）和逆转录PCR（RT—PCR）动态检测不同时间点正常小鼠和白血病小鼠外周血清VEGF及其受体VEGFR-2mRNA表达水平的变化。结果表明：正常小鼠和白血病小鼠均表达VEGF及VEGFR-2mRNA,白血病组外周血清VEGF浓度和VEGFR-2mRNA的表达均显著高于正常组小鼠（P〈0．05）,而且随着病程的进展而逐渐升高。结论：VEGF及VEGFR-2在急性髓系白血病中存在有异常表达,对AML的发生和发展可能起一定的作用。     

姜黄素雷公藤内酯醇对前列腺癌细胞PC3及血管内皮生长因子影响的比较

目的研究姜黄素（curcumin,Cur）、雷公藤内酯醇（triptolide,TL）对非雄激素依赖性前列腺癌细胞株PC3细胞体外作用及其血管内皮生长因子（vascular endothelial growth factor,VEGF）表达的影响。方法分别用梯度浓度的Cur和TL作用于PC3细胞,MTT法检测细胞生长活性;流式细胞仪测定细胞周期及凋亡的变化;半定量RT-PCR法检测PC3细胞内VEGF mR-NA的表达;ELISA检测细胞上清液中分泌VEGF蛋白的浓度。结果Cur及TL都能呈剂量与时间依赖性显著抑制PC3细胞的生长,不同浓度组之间及不同作用时间组之间的差异均有统计学意义（P〈0.01）。Cur、TL诱导PC3细胞分别出现剂量依赖性G2/M、S期阻滞（P〈0.01）,且各浓度组凋亡细胞比例差异有统计学意义（P〈0.01）;PC3细胞内VEGF mRNA的表达和细胞上清液中分泌VEGF蛋白亦呈剂量依赖性降低。结论Cur、TL能显著抑制体外PC3细胞的生长,分别促进细胞周期阻滞于不同时期,增加凋亡,并且VEGF mRNA及蛋白的表达明显降低,两药抑制肿瘤和血管生长机制不同。     

脂多糖诱导人支气管上皮细胞β防御素-3表达的时效和量效性

目的观察不同浓度脂多糖（LPS）诱导人支气管上皮细胞（HBEC）不同时间8防御素-3（hBD-3）的表达,探索hBD-3在呼吸道感染中的作用及与细菌感染的量效关系、时效关系,为人工调控其分泌、防治呼吸道感染进行基础研究。方法不同浓度LPS（0．01、0．1、1、10mg／L）诱导HBEC后,分别在不同时间点（1、2、4和8h）用RT—PCR法检测hBD-3mRNA的表达;同时用0．1mg／LLPS诱导HBEC4h后采用免疫细胞化学检测hBD-3蛋白的表达。结果0．1mg／LLPS诱导HBEC4h后,可见培养的支气管上皮细胞胞质呈棕褐色阳性颗粒,hBD-3蛋白明显表达;不同浓度LPS诱导细胞相同时间后,在同一时间点随浓度增加hBD-3mRNA表达亦随之增加,不同浓度组间比较差异有统计学意义（P〈0．01）;相同浓度LPS在诱导细胞不同时间后,在同一浓度组hBD-3mRNA表达亦随时间而增加,不同时间组间比较差异有统计学意义（P％0．01）。结论hBD-3在HBEC胞质有表达,LPS能诱导HBEChBD-3表达上调,LPS诱导hBD-3表达在一定范围内呈剂量和时间依赖性,hBD-3可能参与气道对LPS最初的防御反应。     

普伐他汀对激素性坏死股骨头内血管内皮细胞生长因子表达的影响

目的 研究普伐他汀对激素性坏死股骨头内血管内皮细胞生长因子（VEGF）表达的影响。方法 54只新西兰白兔随机分为正常组（A组）、模型组（B组）和普伐他汀治疗组（C组），每组18只。B、C2组应用大肠杆菌内毒素及甲泼尼龙琥珀酸钠造成激素性股骨头坏死模型，C组于造模后4周开始应用普伐他汀灌胃，A、B2组灌服等容量的蒸馏水。分别于喂药后4、8、12周分批处死动物，实时定量逆转录聚合酶链反应检测股骨头内VEGF mRNA的表达。结果 B、C2组各期VEGF的mRNA表达水平均低于A组，但C组喂药后第8、12周VEGF mRNA的表达量明显高于B组，差异有显著性（P〈0．01）。结论 普伐他汀可诱导激素性坏死股骨头内VEGF mRNA表达的上调，有可能成为临床治疗激素性股骨头坏死的有效药物。     

耐力运动对雌激素依赖性乳腺癌小鼠肿瘤血管生成的影响

摘要 目的：探讨耐力运动对雌激素依赖性乳腺癌肿瘤血管生成的影响。 方法：40只雌性BALB/c小鼠随机分为2组,一组小鼠在跑台上进行8周持续耐力运动,另一组不进行运动。之后给予2组小鼠注射雌激素依赖性MC4L2癌细胞,肿瘤形成后,随机再将这两组小鼠以每组10只,分为4组,即：运动-肿瘤-运动组（A）;运动-肿瘤-休息组（B）;休息-肿瘤-运动组（C）和休息-肿瘤-休息组（D）。A、C组小鼠进行为期6周的耐力运动,每周运动5天。期间,每周测量肿瘤体积。最后,处死小鼠,摘除肿瘤组织,制成匀浆ELISA定量法测量细胞因子白细胞介素-6（IL-6）和血管内皮生长因子（VEGF）的表达水平。 结果：A、C组与B、D组相比,IL-6（P=0.001）、VEGF（P=0.0001）和肿瘤体积（P=0.0001）显著降低。 结论：运动可致使乳腺癌细胞致瘤小鼠的肿瘤组织内促炎症细胞因子水平降低,抑制肿瘤血管生成因子表达增加。因此,运动对雌激素依赖的乳腺癌可能有一定的辅助治疗与预防作用。     

冰片与丹酚酸B和三七总皂苷配伍对局灶性脑缺血-再灌注大鼠脑VEGF mRNA表达的影响

目的：探讨冰片及与丹参和三七水溶性组分配伍对缺血性中风大鼠脑血管内皮生长因子（VEGF）表达的影响。方法：线栓法复制大鼠大脑中动脉缺血-再灌注损伤模型。动物随机分为模型组、冰片组、丹参三七合剂组（丹七组）和丹参三七合剂加冰片组（丹冰组），各组于再灌注后24、48和72h灌胃给药，分别给予质量分数为0．5％的羧甲基纤维素钠（CMC—Na）、冰片和CMC—Na混悬液、丹参三七合剂（丹酚酸B与三七总皂苷组分组成）、丹参三七合剂加冰片，以正常大鼠为对照组，每组大鼠分别于再灌注24、48和72h末次给药后1h处死取脑。逆转录-聚合酶链反应（RT—PCR）法检测缺血侧脑组织VEGF mRNA表达。结果：再灌注72h冰片可使VEGF mRNA表达明显上调；冰片与丹参三七配伍后，可使再灌注72hVEGF mRNA表达上调。结论：冰片及丹参三七配伍后可通过促进VEGF的表达发挥脑保护作用，单纯冰片对损伤修复阶段（再灌注72h）VEGF表达具有明显诱导作用，与丹参三七组分配伍后，对增强损伤严重阶段（再灌注48h）的抗损伤能力作用显著。     

不同葡萄糖浓度对体外培养肝细胞内caspase-3表达的影响

[目的]探讨不同葡萄糖浓度下肝细胞的caspase-3表达及意义.[方法]将不同浓度的葡萄糖（5、15、25 mol/L）作用于正常成人肝细胞HL-7702 24 h和48 h后,采用流式细胞术检测肝细胞的凋亡情况,采用western blotting法检测肝细胞caspase-3及cleavage caspase-3的表达情况,采用Real-time PCR 检测Bax和Bcl-2蛋白的表达情况.[结果]肝细胞在葡萄糖处理后呈现剂量和时间依赖性的凋亡上调;肝细胞中caspase-3及cleavage caspase-3表达阳性指数也随着葡萄糖浓度和时间逐渐增加;Bax mRNA呈浓度和时间依赖性的增加,而Bcl-2 mRNA的表达水平呈葡萄糖浓度和时间依赖性的下降.[结论]高浓度葡萄糖可诱导干细胞出现凋亡,其作用机制可能通过下调Bcl-2/Bax比值以及caspase-3的剪切活化来实现.     

二烯丙基二硫化物对HL-60白血病细胞株VEGF表达及分泌的影响

为了研究二烯丙基二硫化物（diallyl disulfide，DADS）对白血病HL-60细胞株VEGF mRNA的表达及VEGF蛋白分泌的作用，并从影响VEGF生成的角度探讨DADS的抗白血病机制，应用ELISA法检测药物作用前后白血病HL-60细胞培养上清液中VEGF蛋白的含量；RT-PCR法检测药物作用前后白血病HL-60细胞VEGF mRNA的相对含量。结果表明：HL-60细胞中均有VEGF mRNA的表达和VEGF蛋白的分泌；与空白对照相比，DADS3个浓度组（0，625，1，25，2，5μg／ml）作用HL-60细胞48小时和72小时均能下调HL-60细胞VEGF mRNA的表达及VEGF蛋白的分泌（P〈0.01）。3个浓度组间差异显著（P〈0．01），其下调HL-60细胞VEGF mRNA的表达及VEGF蛋白的分泌效果与浓度相关（r〉0.9，P〈0．01）。结论：DADS可能通过抑制VEGF mRNA的表达及VEGF蛋白的分泌发挥抗白血病效应。     

高糖对脐静脉内皮细胞血管内皮生长因子mRNA表达的影响

目的 观察高糖对脐静脉血管内皮细胞生长因子(VEGF)信使核糖核酸(mRNA)表达的影响.方法 胰酶消化法制备人脐静脉内皮细胞并传代培养,取生长良好的第2、3代细胞进行实验.用不同浓度葡萄糖(0 mmol/L、5.5mmol/L、11 mmol/L、22 mmol/L、44 mmol/L)作用48 h,并选取22 mmol/L葡萄糖分别作用0 h、24 h、48 h、72 h、96 h,观察VEGF mRNA的变化.结果 脐静脉VEGF mRNA的表达随高糖浓度的增加也逐渐增加(P＜0.05),至22 mmol/L达到最高峰,44 mmol/L时下降;在22 mmol/L葡萄糖作用下,随着作用时间的延长,VEGF mRNA水平逐渐增加(P＜0.01),但当作用72 h时VEGF mRNA水平不再升高.结论 高血糖本身可引起脐静脉内皮细胞VEGF mRNA表达的增加,且在一定范围内呈时间和剂量依赖性.     

Mast Cells Can Secrete Vascular Permeability Factor/ Vascular Endothelial Cell Growth Factor and Exhibit Enhanced Release after Immunoglobulin E–dependent Upregulation of Fcε Receptor I Expression

Vascular permeability factor/vascular endothelial cell growth factor (VPF/VEGF) can both potently enhance vascular permeability and induce proliferation of vascular endothelial cells. We report here that mouse or human mast cells can produce and secrete VPF/VEGF. Mouse mast cells release VPF/VEGF upon stimulation through Fcε receptor I (FcεRI) or c-kit, or after challenge with the protein kinase C activator, phorbol myristate acetate, or the calcium ionophore, A23187; such mast cells can rapidly release VPF/VEGF, apparently from a preformed pool, and can then sustain release by secreting newly synthesized protein. Notably, the FcεRI-dependent secretion of VPF/VEGF by either mouse or human mast cells can be significantly increased in cells which have undergone upregulation of FcεRI surface expression by a 4-d preincubation with immunoglobulin E. These findings establish that at least one cell type, the mast cell, can be stimulated to secrete VPF/VEGF upon immunologically specific activation via a member of the multichain immune recognition receptor family. Our observations also identify a new mechanism by which mast cells can contribute to enhanced vascular permeability and/or angiogenesis, in both allergic diseases and other settings.     

Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9-mediated progenitor cell mobilization

Mast cells accumulate in tissues undergoing angiogenesis during tumor growth, wound healing, and tissue repair. Mast cells can secrete angiogenic factors such as vascular endothelial growth factor (VEGF). Ionizing irradiation has also been shown to have angiogenic potential in malignant and nonmalignant diseases. We observed that low-dose irradiation fosters mast cell-dependent vascular regeneration in a limb ischemia model. Irradiation promoted VEGF production by mast cells in a matrix metalloproteinase-9 (MMP-9)-dependent manner. Irradiation, through MMP-9 up-regulated by VEGF in stromal and endothelial cells, induced the release of Kit-ligand (KitL). Irradiation-induced VEGF promoted migration of mast cells from the bone marrow to the ischemic site. Irradiation-mediated release of KitL and VEGF was impaired in MMP-9-deficient mice, resulting in a reduced number of tissue mast cells and delayed vessel formation in the ischemic limb. Irradiation-induced vasculogenesis was abrogated in mice deficient in mast cells (steel mutant, Sl/Sl(d) mice) and in mice in which the VEGF pathway was blocked. Irradiation did not induce progenitor mobilization in Sl/Sl(d) mice. We conclude that increased recruitment and activation of mast cells following irradiation alters the ischemic microenvironment and promotes vascular regeneration in an ischemia model. These data show a novel mechanism of neovascularization and suggest that low-dose irradiation may be used for therapeutic angiogenesis to augment vasculogenesis in ischemic tissues.     

T cell-dependent mast cell degranulation and release of serotonin in murine delayed-type hypersensitivity

We have previously suggested that the release of serotonin (5- hydroxytryptamine) (5-HT) by local tissue mast cells is required for the elicitation of delayed-type hypersensitivity (DTH) in mice. In the current study, light microscopic radioautographs from animals treated with [3H]5-HT indicated that local mast cells released 5-HT between 6 and 18 h during the evolution of DTH. Ultrastructural examination of mast cells revealed surface activation, indicated by extension of surface filopodia, and degranulation by fusion and exocytosis. Light and electron microscopic studies of the endothelium of postcapillary venules at sites of DTH revealed the development of gaps between adjacent cells. The development of gaps permitted extravasation of tracers that was abolished by depletion or antagonism of 5-HT. Thus mast cells degranulated and released 5-HT in DTH, and this 5-HT acted on local vessels. Recipients of nonadherent, non-immunoglobulin-bearing sensitized lymphocytes also demonstrated similar mast cell degranulation and the formation of endothelial gaps. This indicated that mast cell degranulation and 5-HT release in murine DTH were probably T cell dependent.     

Reevaluation of reserpine-induced suppression of contact sensitivity. Evidence that reserpine interferes with T lymphocyte function independently of an effect on mast cells

It has been suggested that reserpine blocks expression of delayed hypersensitivity (DH) by depleting tissue mast cells of serotonin (5-HT), thereby preventing a T cell-dependent release of mast cell 5-HT necessary to localize and to amplify the DH response. However, reserpine blocks expression of DH in mast cell-deficient mice. We therefore decided to reevaluate the mechanism by which reserpine abrogates expression of cellular immunity, and investigated whether the drug might interfere with T cell activity in vitro or in vivo. At concentrations as low as 4 microM, reserpine profoundly suppressed baseline or antigen-augmented levels of [3H]thymidine incorporation by immune lymph node cells obtained from mice sensitized to the contactant oxazolone [I-LNC(Ox)]. This effect was observed both with I-LNC derived from normal mice and with I-LNC derived from congenitally mast cell-deficient W/Wv mice, cell preparations that lacked detectable mast cells, histamine, and 5-HT. Furthermore, treatment of I-LNC with reserpine (20 microM) for 1 h in vitro virtually abolished the ability of these cells to transfer CS to naive mice. This was not a cytolytic effect, as the viability of the I-LNC treated with reserpine was not affected, and washing of the reserpine-treated I-LNC before transfer fully restored their ability to orchestrate a CS response. The action of the drug was not mediated by an effect on mast cells, since the experiment could be performed using mast cell-deficient W/Wv mice as both donors and recipients of I-LNC. In addition, the effect was specific for the treated cells: mice that received reserpine-treated I-LNC(Ox) intravenously together with untreated I-LNC(DNFB) did not develop CS to Ox but responded normally to DNFB; and local intradermal injection of reserpine-treated I-LNC(Ox) which failed to transfer reactivity to Ox, did not interfere with the development of CS to DNFB at the same site. Finally, cotransfer experiments indicated that the effect of reserpine on the transfer of CS was not due to activation of suppressor cells. Our findings strongly suggest that whatever effects reserpine might have on immunologically nonspecific host cells, the drug's effects on sensitized T cells are sufficient to explain its ability to block cell-mediated immune responses in vivo.     

Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the Fc epsilon RI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor alpha

Chronic allergic diseases and other disorders associated with mast cell activation can also be associated with tissue fibrosis, but a direct link between mast cell mediator release and fibroblast collagen gene expression has not been established. Using in situ hybridization, we show that the elicitation of an IgE-dependent passive cutaneous anaphylaxis (PCA) reaction in mice results in a transient, but marked augmentation of steady state levels of type alpha-1 (I) collagen mRNA in the dermis. While peak levels of collagen mRNA expression in the skin are observed 16-24 h after mast cell activation, substantial numbers of dermal cells are strongly positive for collagen mRNA at 1 and 2 h after antigen challenge, before circulating inflammatory cells are recruited into the tissues. Furthermore, experiments in mast cell- reconstituted or genetically mast cell-deficient WBB6F1-W/Wv mice demonstrate that the increased expression of collagen mRNA at sites of PCA reactions is entirely mast cell dependent. In vitro studies show that the supernatants of mouse serosal mast cells activated via the Fc epsilon RI markedly increase type alpha-1 (I) collagen mRNA levels in mouse embryonic skin fibroblasts, and also upregulate collagen secretion by these cells. The ability of mast cell supernatants to induce increased steady state levels of collagen mRNA in mouse skin fibroblasts is markedly diminished by absorption with antibodies specific for either of two mast cell-derived cytokines, transforming growth factor beta (TGF-beta 1) or tumor necrosis factor alpha (TNF- alpha), and is eliminated entirely by absorption with antibodies against both cytokines. Taken together, these findings demonstrate that IgE-dependent mouse mast cell activation can induce a transient and marked increase in steady state levels of type alpha-1 (I) collagen mRNA in dermal fibroblasts and that mast cell-derived TGF-beta 1 and TNF-alpha importantly contribute to this effect.     

Effects of clarithromycin on inflammatory cell mediator release and survival

BACKGROUND: Clarithromycin exhibits anti-inflammatory as well as antimicrobial activity, leading to decreased symptoms of asthma and chronic sinusitis. The mode of anti-inflammatory effects of clarithromycin on inflammatory cells is not well understood. We hypothesized that clarithromycin inhibits inflammatory cell mediator release and survival. METHODS: We investigated the effects of this drug on survival and mediator release from mast cells, eosinophils and neutrophils. RESULTS: Human eosinophil and neutrophil respiratory burst was inhibited by up to 54% after 1-2 h pretreatment with 100 microg/ml clarithromycin. Similar doses of erythromycin did not affect respiratory burst responses in these cells. Clarithromycin at doses of up to 100 microg/ml had no effect on granule-derived mediators released from mast cells and neutrophils. However, we found that clarithromycin (100 microg/ml) induced cell death in mast cells and eosinophils after 16-48 h incubation. CONCLUSION: Clarithromycin inhibited inflammatory cell mediator release and survival, which may enhance its ability to reduce the symptoms of chronic sinusitis and asthma.     

Effects of dexamethasone on mediator release from human lung fragments and purified human lung mast cells.

Purified human lung mast cells released histamine, leukotrienes, prostaglandin (PG) D2, thromboxane B2 (TxB2), and PGF2 alpha in response to anti-IgE stimulation. Incubation of the cells for 24 h with 10(-6) M dexamethasone, a treatment that inhibits mediator release from human basophils, had no effect on the release of these mediators from mast cells. Dexamethasone treatment of human lung fragments led to little or no inhibition of anti-IgE-induced release of the mast cell-derived mediator, histamine, but produced a significant inhibition of the release of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. As was the case with purified mast cells, the steroid did not inhibit the release of PGD2 or TxB2 from human lung fragments. Comparison of the quantities of PGD2 and TxB2 produced by purified cells and human lung fragments reveals that the mast cells produce quantities of these metabolites sufficient to account for the entire amount produced by challenged lung fragments. Dexamethasone inhibited spontaneous release from lung fragments of all cyclooxygenase products measured. These results suggest that the human lung parenchymal mast cell phospholipase is not inhibited by dexamethasone, whereas other phospholipase(s) in the lung are inhibited by the steroid. These results may be useful in explaining the resistance of acute allergic reactions, including anaphylaxis, to steroids, despite the potent antiinflammatory activity of steroids on subacute and chronic inflammation, such as in bronchial asthma, which may be initiated by IgE-dependent mechanisms.     

Modulation of macrophage superoxide-induced cytochrome c reduction by mast cells

The effect of mast cells and mast cell granules on macrophage O2- release as determined by cytochrome c reduction was studied. In vitro activation of mast cells before macrophage activation caused a decrease in O2- -mediated cytochrome c reduction. This decrease was proportional to mast cell activation and reached 80% to 100% when mast cell mediator release was 40% to 50%. Incubation of isolated mast cell granules with macrophages before activation also inhibited O2- -mediated cytochrome c reduction in a dose-dependent manner. Mast cell granule-mediated inhibition of cytochrome c reduction was not caused by histamine, serotonin, or any other dialyzable components but was found to be caused by the scavenging of O2- by mast cell granule-bound superoxide dismutase. Macrophage uptake of sulfur 35-labeled mast cell granules, electron microscopic localization of mast cell granules in the macrophage phagosomes, and the abrogation of mast cell granule effect when the cells were preincubated at 0 degree C indicate that the effect was associated with the adherence or phagocytosis (or both) of mast cell granules. These results suggest that mast cell granules interact with macrophages and that granule superoxide dismutase scavenges O2- generated by the phagocytes.