NGF对哮喘小鼠气道阻力和肺组织Akt/PKB表达的影响

目的探讨NGF介导的Akt/PKB信号转导通路在哮喘小鼠发病中的作用。方法BALB/c小鼠30只,按随机数字表法随机分为正常对照组、哮喘组、NGF阻断组。利用An iRes2005肺功能仪测小鼠气道阻力,运用免疫组织化学方法测定Akt/PKB的组织表达,M etamoph图像分析系统对结果进行分析。结果哮喘小鼠吸气阻力和呼气阻力明显高于正常组小鼠(P<0.01),NGF阻断组小鼠吸气阻力和呼气阻力明显低于哮喘组。免疫组织化学染色结果显示哮喘组Akt/PKB在肺组织炎性细胞的表达及AK t/PKB阳性炎症细胞数明显多于正常对照组(P<0.05),而NGF阻断组则明显低于哮喘组(P<0.05)。结论NGF介导哮喘气道高反应和炎症反应,Akt/PKB参与了哮喘发病中NGF介导的信号传导。     

Akt对哮喘小鼠下呼吸道及内脏感觉传入部位IL-1β表达的影响

目的探讨在哮喘发病机制中,PKB/Akt对哮喘小鼠肺组织及C7-T5段脊神经节和相应节段脊髓后角IL-1β表达的调节作用。方法BALB/c小鼠30只,按随机数字表法均分为正常对照组、哮喘组、PKB/Akt阻断组,免疫组织化学检测各组小鼠肺组织及C7-T5节段脊神经节和相应节段脊髓后角IL-1β的表达,Western blot方法检测各组小鼠肺组织及C7-T5节段脊神经IL-1β的表达。结果免疫组织化学结果显示,哮喘组肺、C7-T5段脊神经节和相应节段脊髓后角IL-1β阳性产物的平均光密度值(MOD)显著高于正常对照组(P<0.01);而PKB/Akt阻断组与哮喘组相比MOD值明显降低(P<0.05)。Western blot结果显示:与正常对照组比较,哮喘组小鼠肺、C7～T5段脊神经中IL-1β的IDV(integrated density value)与β-actin IDV的比值均明显升高(P<0.01),而PKB/Akt阻断组明显低于哮喘组(P<0.05)。结论NGF介导的Akt通路的激活可上调IL-1β的表达,介导哮喘气道高反应和炎症反应。     

高氧对新生鼠肺组织VEGF蛋白表达及肺血管内皮细胞超微结构的影响

目的探讨高浓度氧对新生鼠肺组织血管内皮生长因子(VEGF)蛋白表达及肺血管内皮细胞超微结构影响的动态变化规律。方法建立高浓度氧诱导新生鼠CLD模型,60只新生鼠随机分为实验组和对照组,分别采用免疫组化和透射电镜技术,测定实验组和对照组在生后1d、3d、7d、14d和21dl肺组织内VEGF蛋白表达,同时观察肺血管内皮细胞超微结构变化。结果对照组肺组织VEGF蛋白表达1d主要以传导气道上皮为主,3d以后远端气道上皮表达增加,7d以后肺泡上皮和肺泡间隔明显增多,14d达高峰,以后持续高表达,实验组肺组织VEGF蛋白表达水平7d开始下降,14d以后未见阳性表达。高氧可引起肺血管内皮细胞肿胀、线粒体肿胀和毛细血管基底膜厚薄不均等各种损伤性形态变化,损伤程度随高氧时间延长而加重。结论肺血管的生长是正常肺泡发育重要环节,推测肺组织VEGF蛋白表达下降和肺血管内皮细胞的损伤在高氧诱导CLD肺血管发育障碍中可能发挥重要作用。     

Wortmannin对急性肺损伤小鼠肺组织VEGF和NF-кB表达的影响

目的研究Wortmannin对急性肺损伤模型小鼠肺组织血管内皮生长因子(VEGF)和核转录因子-кB(NF-кB)表达的影响。方法 30只昆明小鼠随机分为正常对照组、急性肺损伤组和Wortmannin处理组。采用腹腔注射LPS(10 mg/kg)建立小鼠急性肺损伤模型,对照组腹腔注射同体积的生理盐水,Wortmannin处理组则于造模前2 h腹腔注射Wortmannin(1.4 mg/kg)。LPS注射后6 h处死大鼠,计算肺组织湿/干重(W/D)比值,Western blot方法检测三组小鼠肺组织内VEGF和NF-кB的表达变化,RT-PCR方法检测三组小鼠肺组织内VEGF mRNA和NF-кB mRNA的表达变化。结果急性肺损伤组小鼠肺组织VEGF和NF-кB蛋白和mRNA表达产物的平均光密度值显著高于正常对照组,而Wortmannin处理组显著低于急性肺损伤组小鼠。结论 Wortmannin能抑制急性肺损伤小鼠肺组织VEGF和NF-кB表达。     

白介素1β上调肺腺癌细胞系A549 VEGF的表达

目的 探讨IL-1β调节肺腺癌细胞A549血管内皮生长因子(VEGF)表达的机制.方法 用不同浓度的IL-1β作用于A549细胞不同时间,再用不同浓度的塞来昔布干预IL-1β诱导的A549细胞,用反转录-聚合酶链反应(RT-PCR)测定VEGF mRNA表达,用ELISA法测定培养上清液中VEGF蛋白质表达.结果 IL-1β干预A549细胞后VEGF mRNA和蛋白质表达均较对照组显著增加,呈剂量依赖性(P＜0.05),24 h时达高峰.不同浓度的塞来昔布显著缓解上述变化(P＜0.05).结论 缓解Il-1β诱导的A549细胞VEGF表达,其调节机制可能发生在转录水平.     

甲状腺癌组织中VEGF和VEGF-C的表达及意义

目的　探讨血管内皮生长因子(VEGF)、VEGF- C在甲状腺癌中的表达及其意义。方法　应用免疫组化S P法检测44例甲状腺癌中VEGF、VEGF C的表达情况,并以17例癌旁正常甲状腺组织作对照。结果　VEGF、VEGF- C在甲状腺癌中呈高水平表达(88. 6%、81. 8% )。VEGF表达随癌组织分化程度的减低而增高, 9例死亡病例均为阳性表达;VEGF- C表达随癌组织分化程度的减低而减低,乳头状癌阳性表达(88 .9% )高于其它类型,VEGF- C阳性率在有淋巴结转移组(92. 0% )明显高于无淋巴结转移组(68. 4% ) (P<0. 05)。9例死亡病例中7例为阳性表达,且7例同时VEGF呈阳性表达。结论　VEGF、VEGF- C表达与甲状腺癌病理分型及预后可能有一定关系,VEGF- C与甲状腺癌淋巴结转移密切相关。     

地塞米松抑制哮喘模型大鼠肺组织血管内皮生长因子表达

哮喘是一种由多种细胞和细胞组分参与的慢性气道炎症性疾病,反复炎症刺激可导致气道高反应性和气道重塑,气道细胞合成和分泌的血管内皮生长因子(vascular endothe-lial growth factor,VEGF)参与了哮喘气道重塑过程,但对哮喘急性发作期VEGF的变化及影响因素的研究却鲜见报道。本     

肺腺癌中VEGF家族及其受体表达与FDG摄取程度的关系

目的研究肺腺癌中血管内皮生长因子D及其受体表达水平与病灶氟代脱氧葡萄糖(FDG)摄取程度的关系,探讨FDG摄取程度是否可作为肺腺癌中预测靶向治疗疗效的一项指标。方法使用寡DNA基因芯片技术(Genespring 7.3)对6例肺腺癌病例行基因水平分析,并使用免疫组化方法对基因分析进一步证实。使用抗VEGF-D抗体对49例肺腺癌病理石蜡包埋切片行免疫组化染色。所有患者常规行PET检查,感兴趣区的定量分析采用标准摄取值(SUV)。使用独立样本t检验,χ2检验及F检验行统计学分析,使用Kaplan-Meier方法计算无病生存曲线。结果 VEGF-D表达在低FDG摄取组中远远高于高FDG摄取组(=0.0241)。免疫组化研究进一步证实:VEGF-D阴性组的FDG摄取程度明显高于阳性组(0.001)。VEGF-D高表达组中发生淋巴结转移(0.001)及复发(0.001)的病例数均明显低于VEGF-D低表达组。VEGF-D高表达的患者其无病生存率明显高于低表达者。结论 VEGF-D表达与肺腺癌患者FDG摄取程度呈负相关,且可能作为肺腺癌患者淋巴结转移,组织学分型及复发的预测指标。PPPP     

五味子哮喘汤对哮喘小鼠SIRT1/Akt信号通路及肺功能的影响

目的:探究五味子哮喘汤(Schisandrae-asthma decoction)对哮喘小鼠沉默信息调节因子1(silent in-formation regulator 1,SIRT1)/蛋白激酶B(protein kinase B,PKB/Akt)信号通路及肺功能的影响.方法:BALB/c小鼠随机分为对照组、模型组...     

Akt/PKB异常与消化系统肿瘤的研究进展

Akt/PKB信号通路是细胞内信号传导的重要环节,参与腚细胞代谢、生存/凋亡、分化和增殖等过程.细胞内Akt/PKB活性状态依赖于其磷酸化与去磷酸化之间的平衡.蛋白磷酸酶PP2A使Akt/PKB去磷酸化而失活,而Ser/Thr蛋白磷酸酶抑制剂冈田酸(okadaic acid)可以增加活化的Akt/PKB.Akt/PKB信号通路调节异常可见于肿瘤、糖尿病甚至精神分裂症等.肿瘤组织中Akt/PKB活性水平高可以是Akt/PKB信号通路上游调节异常造成的,如P13K亚基或:PTEN的基因突变,或者Akt/PKB基凶扩增造成的.Akt/PKB被认为参与了多种肿瘤的发生、发展及侵袭转移.近年Akt/PKB信号通路正逐渐成为肿瘤发牛机制及治疗的研究热点,本文就Akt/PKB异常与消化系统肿瘤的研究进展作一综述.     

神经生长因子对哮喘豚鼠内脏感觉传入部位MMP-2表达的影响

目的探讨基质金属蛋白酶-2(MMP-2)在哮喘豚鼠内脏感觉传入部位的表达及神经生长因子(NGF)对MMP-2的调节作用。方法应用免疫组织化学方法检测豚鼠内脏感觉传入部位MMP-2免疫反应的变化。用W estern b lot蛋白印迹方法检测豚鼠C7~T5段脊神经节和相应节段脊髓背角NGF和MMP-2的表达。用Luzex-F实时图像分析系统和凝胶成像分析系统分别对结果进行图像分析。结果①免疫组织化学结果显示:豚鼠C7~T5段脊神经节和相应节段脊髓背角内MMP-2阳性免疫反应产物的平均灰度值(0-深色,255-浅色),生理盐水组(175.50±4.67、166.47±6.20)与单纯致敏组(172.63±9.51、165.20±5.14)比较没有显著差异(P>0.05);哮喘组(139.87±4.88、125.93±4.49)明显低于生理盐水组和单纯致敏组(P<0.01);哮喘+NGF抗体组(鼻腔吸入NGF抗体,169.73±6.24、152.37±8.67)则明显高于哮喘组(P<0.01)。②W estern b lot结果显示:与生理盐水组和单纯致敏组比较,哮喘组豚鼠C7~T5段脊神经节及相应节段脊髓样品中MMP-2和NGF阳性产物的IDV(Integrated Density Value)与β-actin IDV的比值均明显升高(P<0.01),而哮喘+NGF抗体组则明显低于哮喘组(P<0.01),表明NGF可上调豚鼠内脏感觉传入部位MMP-2的表达。结论C7~T5段脊神经节以及相应节段脊髓背角内的MMP-2可能也参与哮喘的发病过程,NGF诱发MMP-2的表达可能是NGF参与哮喘发病的机制之一,NGF抗体抑制哮喘时MMP-2的表达有可能延缓气道重塑的发生,可能是治疗哮喘的新途径。     

机械张应变诱导蛋白激酶B活化对血管平滑肌细胞迁移的影响

目的研究机械张应变诱导蛋白激酶B活化对大鼠血管平滑肌细胞迁移能力的影响。方法应用FX-4000T细胞应变加载系统,对大鼠血管平滑肌细胞施加牵拉幅度为15%、频率为1Hz的张应变。以Transwell和Westernblot等方法观察张应变作用下蛋白激酶B磷酸化和血管平滑肌细胞迁移能力的变化,以未加载张应变的血管平滑肌细胞为对照组。结果与对照组相比,机械张应变增加细胞中蛋白激酶B磷酸化水平,促进血管平滑肌细胞的迁移;PI3K的特异性抑制剂Wortmannin抑制张应变诱导的蛋白激酶B的磷酸化,降低了血管平滑肌细胞迁移能力。结论机械张应变通过上调蛋白激酶B磷酸化水平促进了血管平滑肌细胞迁移,提示蛋白激酶B信号通路参与机械张应变条件下血管平滑肌细胞迁移过程的信号传导。     

Overexpression of Akt/PKB modulates N‐myristoyltransferase activity in cancer cells

N‐myristoyltransferase (NMT) catalyses the myristoylation reaction. Since NMT activity is elevated in various cancers and activated Akt/PKB leads to cell survival, we were interested in studying if activation of Akt/PKB has any effect on NMT. Overexpression of constitutively active Akt/PKB in HepG2 cells (HepG2‐CA‐Akt/PKB) led to an approximately 50% reduction of NMT compared with parental HepG2 cells. Reduced NMT activity in HepG2‐CA‐Akt/PKB was found to be due to the NMT1 phosphorylation. We determined NMT activity in various human breast cancer cell lines with differing metastatic potentials and pseudo‐normal breast cells (HBL‐100). Tumourigenic or metastatic breast cancer cell lines such as MDA‐MB‐231, MDA‐MB‐435, and Hs 578T displayed reduced NMT activity. Western blot analysis revealed that NMT1 is phosphorylated in these breast cancer cells. Furthermore, patients' breast cancer tissue array revealed strong positivity and high intensity for NMT in malignant breast tissues compared with normal breast cells. A gradation in the NMT staining was observed for grade I, II, and III infiltrating ductal carcinoma breast tissues. These studies demonstrate that overexpression of Akt/PKB results in NMT1 phosphorylation and that NMT1 is phosphorylated in breast cancer cells. Immunohistochemical analysis suggests that NMT may prove to be an added diagnostic biomarker for breast cancer. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

重组人血管内皮抑素联合放疗对Lewis肺癌小鼠肿瘤生长及VEGF表达的影响

目的:研究重组人血管内皮抑素(rh-Endostatin,YH-16,恩度)对不同放射剂量治疗的Lewis肺癌小鼠肿瘤生长及血管内皮生长因子(VEGF)表达的影响。方法:制作Lewis肺癌细胞接种肿瘤的动物模型,并将60只模型小鼠随机分为5组:A,空白对照组(不予任何治疗);B,小剂量多次放疗组(1Gy/f,隔日一次,共4次);C,大剂量单次放疗组(4Gy/f,共1次);D,小剂量多次放疗联合恩度组(放疗前4天起每天右侧腹股沟区皮下注射恩度0.1ml,共15天,放射剂量为1Gy/f,隔日一次,共4次);E,大剂量单次放疗联合恩度组(放疗前4天起每天右侧腹股沟区皮下注射恩度0.1ml,共15天,放射剂量为4Gy/f,共1次)。分别给予上述处理后计算抑瘤率,并用免疫组化SP法测定各组VEGF的阳性表达率。结果:与C、E组比较,B、D组的抑瘤作用及VEGF表达下降明显(P<0.05),尤以D组抑瘤更强(P<0.05)、VEGF下降最多(P<0.05)。结论:放疗前使用恩度可使Lewis肺癌小鼠对放疗的敏感性增加,其机制可能与下调VEGF的表达有关。     

Effects of Diet-Induced Mild Obesity on Airway Hyperreactivity and Lung Inflammation in Mice

Purpose

Obesity has been suggested to be linked to asthma. However, it is not yet known whether obesity directly leads to airway hyperreactivity (AHR) or obesity-induced airway inflammation associated with asthma. We investigated obesity-related changes in adipokines, AHR, and lung inflammation in a murine model of asthma and obesity.

Materials and Methods

We developed mouse models of chronic asthma via ovalbumin (OVA)-challenge and of obesity by feeding a high-fat diet, and then performed the methacholine bronchial provocation test, and real-time PCR for leptin, leptin receptor, adiponectin, adiponectin receptor (adipor1 and 2), vascular endothelial growth factor (VEGF), transforming growth factor (TGF) β, and tumor necrosis factor (TNF) α in lung tissue. We also measured cell counts in bronchoalveolar lavage fluid.

Results

Both obese and lean mice chronically exposed to OVA developed eosinophilic lung inflammation and AHR to methacholine. However, obese mice without OVA challenge did not develop AHR or eosinophilic inflammation in lung tissue. In obese mice, lung mRNA expressions of leptin, leptin receptor, VEGF, TGF, and TNF were enhanced, and adipor1 and 2 expressions were decreased compared to mice in the control group. On the other hand, there were no differences between obese mice with or without OVA challenge.

Conclusion

Diet-induced mild obesity may not augment AHR or eosinophilic lung inflammation in asthma.     

Extracellular HIV-1 Tat protein activates phosphatidylinositol 3- and Akt/PKB kinases in CD4+ T lymphoblastoid Jurkat cells

The biological basis for the pleiotropic activity of extracellular human immunodeficiency virus (HIV)-1 Tat protein on lymphoid T cell survival is not well understood. We have here demonstrated that the addition in culture of 0.1–10 nM Tat protein to 36-h serum-starved lymphoblastoid Jurkat T cells rapidly stimulates the catalytic activity of phosphatidylinositol 3-kinase (PI 3-K). The peak of activation was observed 30 min after Tat addition. Extracellular Tat also stimulated the catalytic activity of the Akt/PKB kinase, a major target of PI 3-K lipid products. Pretreatment of serum-starved Jurkat cells with 100 nM wortmannin (WT) or 10 μM LY294002, two unrelated pharmacological inhibitors of PI 3-K, markedly suppressed the catalytic activity of both PI 3-K and Akt/PKB in Jurkat cells. Moreover, at low concentrations (0.1–1 nM), extracellular Tat showed a small but reproducible protection of Jurkat cells from apoptosis induced by serum deprivation (p < 0.05), while the combination of Tat plus 100 nM WT significantly (p < 0.05) increased the percentage of apoptosis with respect to cells left untreated or treated with Tat alone. Taken together, these data suggest that the anti-apoptotic activity of low concentrations of Tat protein on Jurkat cells is mediated by a PI 3-kinase/Akt pathway.     

DPC4基因转染结肠癌细胞对VEGF表达的影响

目的研究DPC4基因转染人结肠癌细胞株SW620对血管内皮生长因子(VEGF)表达的影响.方法利用DNA的限制性内切酶双酶切技术鉴定重组质粒PCDNA3.1-DPC4;利用脂质体介导转染技术和G418筛选得到稳定表达Smad4蛋白的DPC+4-SW620(PCDNA3.1-DPC4转染的SW620高转移性结肠癌细胞株)细胞模型;采用Western blot检测细胞中Smad4的表达;采用ELISA检测细胞上清液中VEGF的蛋白表达量;采用半定量逆转录多聚酶链反应RT-PCR技术检测细胞中VEGF的mRNA表达量.结果阳性质粒组DPC+4-SW620细胞的Smad4蛋白表达强于空白质粒组PCDNA3.1-SW620和未转染组SW620;DPC+4-SW620组细胞上清液VEGF蛋白分泌明显减少,与PCDNA3.1-SW620组和SW620组比较差异有显著性(P＜0.05),DPC+4-SW620组细胞与PCDNA3.1-SW620组细胞中VEGF mRNA半定量结果和SW620组比较,其表达量分别下降28.3%和4.5%.结论 DPC4可抑制人结肠癌细胞株SW620中VEGF的表达.     

金水鲜胶囊联合放射治疗对 Lewis 肺癌小鼠肿瘤生长和血管内皮生长因子表达影响的研究简

目的观察金水鲜胶囊联合放射治疗（简称放疗）对Lewis肺癌小鼠肿瘤生长及血管内皮生长因子（VEGF）表达的影响。方法建立Lewis肺癌移植瘤小鼠模型,随机分为6组,即空白对照组、金水鲜组、放疗组、先金水鲜后放疗组、先放疗后金水鲜组和同时金水鲜＋放疗组,每组6只。放疗剂量为2Gy,金水鲜制成悬液后灌胃给药。游标卡尺测量瘤体长短径变化。绘制肿瘤生长瞳线：采用实时聚合酶链反应（real-time PCR）和免疫组织化学法检测瘤体VEGF的表达。结果肿瘤体积在接受处理后明显减小。与空白对照组相比,先金水鲜后放疗组、先放疗后金水鲜组和同时金水鲜＋放疗组肿瘤体积减小最为明显（分别为303.209mm^3±14.445mm^3,284.758mm^3±21.891mm^3,303.228mm^3±2.335mm^3）,差异有统计学意义（P〈0.05）。先金水鲜后放疗组、先放疗后金水鲜组和同时金水鲜＋放疗组与空白对照组或放疗组比较,VEGF表达阳性率降低（分别为12.97％±O.82％,15.13％±1.31％。13.62％±0.83％）,差异有统计学意义（P〈0.05）。结论金水鲜胶囊与放疗联用对Lewis肺癌小鼠有抑制肿瘤的作用.其机制可能是下调VEGF的表达。     

Validation of anti-vascular endothelial growth factor (anti-VEGF) antibodies for immunohistochemical localization of VEGF in tissue sections: expression of VEGF in the human endometrium

Transient transfection of COS-1 cells followed by fixation, embedding in paraffin, and immunohistochemistry has identified anti-vascular endothelial growth factor (anti-VEGF) mouse monoclonal antibodies that efficiently immunostain VEGF in paraffin-embedded tissue sections. Immunohistochemical localization of VEGF in 34 specimens of normal human endometrium that had been collected at different stages of the menstrual cycle was then performed. VEGF was present at all stages of the cycle, but both the pattern and the intensity of staining varied. Thus, VEGF expression occurred predominantly in the endometrial epithelium and while weak in the proliferative phase, was strong in the secretory phase. VEGF expression in the stroma was weaker than in the proliferative phase glands and did not change throughout the cycle. These findings are in agreement with reports of VEGF mRNA expression in the endometrium, but disagree with previous immunohistochemical studies that employed an immunohistochemically unvalidated antiserum. This study has shown that the commercially available anti-VEGF monoclonal antibody M293 is excellent for the immunohistochemical localization of VEGF in paraffin sections. © 1998 John Wiley & Sons, Ltd.