Differential activation of host cell signalling pathways through infection with two variants of influenza A/Puerto Rico/8/34 (H1N1) in MDCK cells

In cell culture-based influenza vaccine production, few efforts have been undertaken to characterise virus–host cell interactions in detail. Two influenza virus strains that grew to different virus titres, and differed in virus dynamics, apoptosis induction and proteome changes were observed.     

姜黄素抑制人肝癌细胞BEL-7402中VEGF和HIF-1α表达通过PI3K／AKT／FRAP信号传导通路

目的探讨MAPK和PI3K信号传导通路在姜黄素调节VEGF和HIF-1α表达中的作用。方法分别加入LY29400225μmol／L、50μmol／L，U012610μmol／L、20μmol／L，rapamycin 5ng／ml、10ng／ml处理人肝癌细胞BEL-7402，30min后加入姜黄素10μmol／L，对照组单独加入0、10μmol／L姜黄素，缺氧环境中培养6h后，行RT—PCR和Western Blot检测VEGF蛋白、mRNA和HIF-1α蛋白表达；分别加入不同浓度的的姜黄素以及LY294002 25 μmol／L处理人肝癌细胞BEL-7402，缺氧环境中培养6h后，行Western Blot检测磷酸化AKT和总AKT蛋白表达。结果姜黄素10μmol／L＋LY29400225μmol／L组、姜黄素10μmol／L＋LY294002 50 μmol／L组、姜黄素10μmol／L＋rapamycin 5 ng／ml组、姜黄素10μmol／L＋rapamycin 10 ng／na组HIF-1α蛋白、VEGF蛋白、mRNA表达分别与姜黄素10μmol／L组相比降低，差异有统计学意义（P〈0．01）；而姜黄素10μmol／L＋U012610μmol／L组、姜黄素10μmol／L＋U0126 20 μmol／L组HIF—1α蛋白、VEGF蛋白、mRNA表达分别与姜黄素10μmol／L组相比差异无统计学意义（P〉0.05）；不同浓度的姜黄素、LY294002 25 μmol／L处理的人肝癌细胞BEL-7402缺氧6h后磷酸化AKT蛋白表达逐渐降低，LY294002 25 μmol／L可以基本阻断磷酸化AKT蛋白的表达，而对总AKT蛋白表达无明显变化。结论姜黄素对人肝癌细胞BEL-7402中HIF-1α蛋白和VEGF的表达通过P13K／AKT／FRAP信号传导通路。     

^60Co治疗机计算机辅助操作系统的原理与实现

本文介绍计算机软件《＾６０Ｃｏ治疗机辅助操作系统》的原理与实现。包括：计算“放射是有关公式；治疗质量控制有关指标及计算公式；系统功能模块；系统操作流程；系统运行环境。     

下调LOX基因对肾癌细胞增殖、凋亡及细胞周期的影响及机制研究

目的 探讨LOX对肾癌细胞增殖、凋亡及细胞周期的影响及其作用机制。方法 将si-con、si-LOX质粒分别转染至786-0细胞中,记为si-con组、si-LOX组,转染采用脂质体法。蛋白质印迹（Western Blot）检测赖氨酰氧化酶(LOX)、周期素依赖性激酶4（CDK4）、Bcl-2相关X蛋白（Bax）、B细胞淋巴瘤/白血病-2（Bcl-2）、磷酸化蛋白激酶B（p-Akt）、蛋白激酶B（Akt）表达水平;四甲基偶氮唑盐比色法(MTT)检测细胞活力;流式细胞术检测细胞凋亡和细胞周期。结果 与正常肾小管上皮细胞HK-2相比,肾癌细胞786-0、GRC-1、A498中LOX表达水平显著升高（P＜0.001）。敲减LOX肾癌细胞786-0的活力显著降低（t=8.440,P＜0.001）,786-0细胞的凋亡率显著升高（t=25.628,P＜0.001）,G0/G1期细胞所占比例增加（t=7.211,P＜0.001）,S期细胞所占比例减少（t=13.190,P＜0.001）,786-0细胞中CDK4、Bcl2、p-Akt表达水平显著降低（t=15.660、13.914、12.349,P＜0.001）,786-0细胞中Bax表达水平显著升高（t=22.057,P＜0.001）。结论 敲减LOX能抑制肾癌细胞增殖,促进细胞凋亡,影响细胞周期,其机制可能与PI3K/Akt信号通路有关,可为肾癌的治疗提供新思路。     

不同n-3/n-6多不饱和脂肪酸构成比膳食对大鼠一磷酸腺苷活化蛋白激酶表达的影响

目的研究不同n-3/n-6配比脂肪酸对大鼠磷酸腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)蛋白及活性表达的影响。方法58只SD大鼠适应性喂养7d后,尾静脉取血。根据血清总胆固醇水平随机分为:空白(基础饲料);高脂(高脂饲料);高脂1:1(高脂饲料+n-3/n-6=1:1配方油);高脂1:5(高脂饲料+n3/n6=1:5配方油);低脂1:1(脱脂基础饲料+n-3/n-6=1:1配方油);低脂1:5(脱脂基础饲料+n3/n6=1:5配方油)6组,喂养45d,观察大鼠摄食与体重增长。于实验前1d,15d,30d,45d分别各取血测血清总胆固醇水平,于D45处死动物。Western blotting分别分析肝和下丘脑组织中AMPK-α总蛋白及其活性表达。结果添加PUFA的4个比例组血清TC、体重与高脂组相比,显著降低,且低脂2个比例组和高脂1:1组均与高脂1:5组相比有显著差异。添加PUFA的4个比例组均与高脂组相比,大鼠下丘脑AMPK-α总蛋白表达水平明显降低,肝AMPK-α蛋白表达水平均比高脂组明显升高。结论PUFA改善血脂可能是通过增加肝AMPK表达,抑制下丘脑AMPK表达,增加肝脂肪酸氧化和抑制食欲,影响血脂代谢。     

Effects of Phosphoethanolamine Supplementation on Mitochondrial Activity and Lipogenesis in a Caffeine Ingestion Caenorhabditis elegans Model

Caffeine intake is strongly linked to lipid metabolism. We previously reported the age-dependent physiological effects of caffeine intake in a Caenorhabditis elegans model. Since nutritional status can actively influence metabolism and overall health, in this study, we evaluated the effect of caffeine intake on lipid metabolism in adult-stage C. elegans. We found that, in C. elegans, fat storage and the level of phosphoethanolamine (PE) were significantly reduced with caffeine intake. In addition, mitochondrial activity decreased and mitochondrial morphology was disrupted, and the expression of oxidative stress response genes, hsp-6, gst-4, and daf-16, was induced by caffeine intake. Furthermore, the level of an energy metabolism sensor, phospho-AMP-activated protein kinase, was increased, whereas the expression of the sterol regulatory element binding protein gene and its target stearoyl-CoA desaturase genes, fat-5, -6, and -7, was decreased with caffeine intake. These findings suggest that caffeine intake causes mitochondrial dysfunction and reduces lipogenesis. Interestingly, these changes induced by caffeine intake were partially alleviated by PE supplementation, suggesting that the reduction in mitochondrial activity and lipogenesis is in part because of the low PE level, and proper dietary supplementation can improve organelle integrity.     

血清及尿液中sFlt-1/PLGF比值的变化与子痫前期相关性研究

目的:研究子痫前期患者及正常对照组孕妇血清及尿液中胎盘生长因子(PLGF)与可溶性络氨酸激酶受体1(sFlt-1)水平变化,探讨血清及尿液中sFlt-1/PLGF比值的变化与子痫前期病情程度的关系,以及尿液中PLGF、sFlt-1水平与血清中PLGF、sFlt-1水平的相关性。方法:采用酶联免疫吸附法检测并比较25例轻度子痫前期患者、20例重度子痫前期患者及20例正常晚期妊娠妇女血清及尿液中sFlt-1/PLGF比值,并分析子痫前期患者及对照组孕妇尿液中sFlt-1、PLGF与血清中sFlt-1、PLGF水平的相关性。结果:对照组血清及尿液中sFlt-1/PLGF值低于轻度子痫前期组,轻度子痫前期组血清及尿液中sFlt-1/PLGF值低于重度子痫前期组,各组之间的差异有统计学意义(P<0.05)。尿液中PLGF、sFlt-1水平与血清中PL-GF、sFlt-1水平成正相关。结论:孕妇血清及尿液中sFlt-1/PLGF比值水平与子痫前期病情程度呈相关性,且尿液中PLGF、sFlt-1水平与血清中PLGF、sFlt-1水平成正相关,可以用尿液中sFlt-1/PLGF比值检测替代血清sFlt-1/PLGF比值预测子痫前期的发生。     

ERK1/2在维生素E琥珀酸酯诱导人胃癌细胞凋亡中的作用

为探讨ERK1 2丝裂素活化蛋白激酶 (MAPK)信号转导途径在维生素E琥珀酸酯 (VES)所诱导的人胃癌SGC 790 1细胞凋亡过程中的作用 ,采用DAPI染色法观察VES诱导细胞凋亡的情况 ,采用WesternBlot法分析VES不同剂量和不同作用时间对ERK1 2磷酸化状态的影响。结果表明 ,VES明显诱导SGC 790 1细胞凋亡 ,2 0 μg mlVES作用 2 4h和 48h后凋亡率分别为 14 .2 %和 89.6%。 5、10、2 0 μg mlVES作用 2 4h时明显降低p ERK蛋白的表达 ;而 2 0 μg mlVES作用于细胞时 ,ERK1 2可瞬时被激活 ,2h时表达下降 ,而后又升高 ,12h达峰值。提示ERK1 2途径可能参与了VES诱导的SGC 790 1细胞凋亡 ,但最终参与调节细胞增殖过程     

Digestion kinetics of potato protein isolates in vitro and in vivo

Abstract

Recently, an industrial process was developed to isolate native protein fractions from potato: a high (HMW) and a low (LMW) molecular weight fraction. Digestion kinetics of HMW and LMW was studied in vitro and in vivo and compared with reference proteins. Under simulated conditions, highest digestion was found for whey protein, followed by soy, pea, HMW, casein and LMW. Ingestion of 20?g of proteins by eight healthy subjects (following a randomized, double-blind, cross-over design) induced a slow and moderate increase with HMW and LMW, while a peaked and high increase with whey protein, in postprandial plasma amino acid levels. Casein gave a similar profile as HMW, with higher levels. Contrary to whey and casein, HMW and LMW did not result in any changes in plasma insulin or glucose levels. This study provides insights in digestion of native potato protein isolates to assist their use as protein sources in food applications.     

Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on Uncoupling Protein 3 Gene Expression in C2C12 Muscle Cells

Uncoupling protein 3 (UCP3) is a mitochondrial membrane transporter that is expressed mainly in skeletal muscle where it plays an important role in energy expenditure and fat oxidation. In this study, we investigated the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on UCP3 gene expression in C₂C₁₂ muscle cells. EPA and DHA up-regulated UCP3 mRNA level in a dose-dependent manner and similarly increased UCP3 promoter activity in C₂C₁₂ muscle cells. To determine whether AMP-activated protein kinase (AMPK) signaling may also directly regulate UCP3 expression, 5′-amino-4-imidazolecarboxamide-ribonucleoside (AICAR), an AMP analog that activates AMPK, was treated in C₂C₁₂ muscle cells. AICAR showed additive effects with EPA or DHA on the UCP3 promoter activation. These results indicate that EPA and DHA directly regulate the gene expression of UCP3, potentially through AMPK-mediated pathway in C2C12 muscle cells.     

ERK通路参与调控胰腺癌细胞吉西他滨化疗抵抗

目的 检测ERK蛋白在胰腺癌细胞株产生吉西他滨（GEM）化疗抵抗前后的表达,分析ERK通路在化疗抵抗中的作用.方法 通过浓度梯度递增诱导法建立对不同浓度吉西他滨化疗抵抗的胰腺癌细胞株模型 应用免疫组化检测各个化疗抵抗浓度细胞株ERK的表达 使用ERK通路阻断剂和激活剂分别作用于化疗抵抗最低和最高浓度,再检测ERK的表达 运用MTT法检测各因素对细胞耐药的影响,计算IC50值.结果 经过吉西他滨化疗诱导后,ERK的表达随着化疗抵抗浓度的增加而升高 ERK通路特异性阻断剂作用于最低及最高浓度的化疗抵抗细胞株后,ERK蛋白的表达降低,细胞的IC50值降低,细胞对化疗药物的敏感性增强 ERK通路激活剂作用后,ERK蛋白的表达增强,细胞的IC50值升高,细胞对化疗药物的敏感性增强.结论 ERK通路参与调控胰腺癌细胞吉西他滨化疗抵抗.     

The Non-Genomic Actions of Vitamin D

Since its discovery in 1920, a great deal of effort has gone into investigating the physiological actions of vitamin D and the impact its deficiency has on human health. Despite this intense interest, there is still disagreement on what constitutes the lower boundary of adequacy and on the Recommended Dietary Allowance. There has also been a major push to elucidate the biochemistry of vitamin D, its metabolic pathways and the mechanisms that mediate its action. Originally thought to act by altering the expression of target genes, it was realized in the mid-1980s that some of the actions of vitamin D were too rapid to be accounted for by changes at the genomic level. These rapid non-genomic actions have attracted as much interest as the genomic actions and they have spawned additional questions in an already busy field. This mini-review attempts to summarise the in vitro and in vivo work that has been conducted to characterise the rapid non-genomic actions, the mechanisms that give rise to these properties and the roles that these play in the overall action of vitamin D at the cellular level. Understanding the effects of vitamin D at the cellular level should enable the design of elegant human studies to extract the full potential of vitamin D to benefit human health.     

Proteomic Analysis of Cellular and Membrane Proteins in Fluconazole-Resistant Candida glabrata

ObjectivesCandida glabrata is one of the most common causes of Candida bloodstream infections worldwide. Some isolates of C glabrata may be intermediately resistant to azoles, with some strains developing resistance during therapy or prophylaxis with fluconazole. In this study, we used a proteomic approach to identify differentially expressed proteins between fluconazole-resistant and -susceptible strains.MethodsMembrane and cellular proteins were extracted from fluconazole-susceptible and fluconazole-resistant C glabrata strains. Differentially expressed proteins were compared using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with >1.5-fold difference in expression were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS).ResultsA total of 65 proteins were differentially expressed in the cellular and membrane fractions. Among the 39 cellular proteins, 11 were upregulated and 28 were downregulated in fluconazole-resistant strains in comparison with fluconazole-susceptible strains. In the membrane fraction, a total of 26 proteins were found, of which 19 were upregulated and seven were downregulated. A total of 31 proteins were identified by LC-MS/MS that are involved in glycolysis, carbohydrate transport, energy transfer, and other metabolic pathways. Heat shock proteins were identified in various spots.ConclusionHeat shock and stress response proteins were upregulated in the membrane fraction of the fluconazole-resistant C glabrata strain. Compared with susceptible strains, fluconazole-resistant strains showed increased expression of membrane proteins and decreased expression of cellular proteins.     

海湾战争综合征模型大鼠海马脑源性神经营养因子及其受体和调节因子的表达

目的探讨脑源性神经营养因子（BDNF）对海湾战争综合征（GWS）海马细胞凋亡或存活的作用及其机制．为应用BDNF预防和／或治疗GWS提供理论依据。方法入选大鼠随机分为对照组、束缚应激组和GWS模型组。实验28d结束后处死动物,取脑。采用免疫组化技术和免疫印迹分析检测海马CAl区BDNF及其受体酪氨酸蛋白激酶B（TrkB）和cAMP反应元件结合蛋白（CREB）的表达。结果束缚应激组大鼠海马BDNF、TrkB及CREB表达的平均吸光度值分别为0．291±0．001、0．253±0．002、0．215±0．013。明显低于对照组（BDNF0．320±0．040、TrkB0．243±0．004、CREB0．232±0．009）（P〈0．05）：GWS模型大鼠海马BDNF、TrkB及CREB表达的平均吸光度值分别为O．269±0．036、0．226±0．004、0．194±0．010,明显低于低于对照组和束缚应激组（P〈0．05）。结论GWS模型大鼠海马CAl区BDNF及其受体TrkB以及调节因子CREB的表达明显下降。     

Age-related changes in memory and effector T cells responding to influenza A/H3N2 and pandemic A/H1N1 strains in humans

Age-dependent changes in the cellular immune response have been mainly described in CD8+ T cells, with relative sparing of CD4+ T cells. We show that in older compared to young adults, effector memory and effector CD8+ T-cell subsets responding to influenza A/H3N2 challenge have diminished cytolytic activity. In contrast, effector CD4+ T-cell subsets in older adults share similar phenotypic and functional characteristics with those from young adults. Further, we observed a diminished cytolytic T-cell response to both seasonal influenza A/H3N2 and pandemic H1N1 (pH1N1) strains in older compared to young adults who had received seasonal influenza vaccine. These results are consistent with the observed rates of serious complications from seasonal and pandemic influenza infections in different age groups, and suggest that CD4+ T cells may provide a compensatory response to influenza infection when CD8+ T cells become compromised during the aging process.     

SLE患者中DcR3的表达及作用机制的研究

目的:探讨DcR3在SLE免疫紊乱过程中发生的变化及其可能作用机制。方法:选择36例SLE患者,以12例健康体检者为对照;采用ELISA法检测血清中的DcR3I、L-4和IFN-γ。结果:SLE组血清DcR3I、L-4和IFN-γ量显著高于正常对照组(P均<0.01);SLE患者中I,FN-γ/IL-4值呈两极化分布特点,以0.76为界,分为较明显的高值组(n=19,1.93±1.14)和低值组(n=170,.53±0.09)I,FN-γ/IL-4低值组的DcR3和IL-4水平比IFN-γ/IL-4高值组和正常对照组均高;DcR3与IL-4呈正相关趋势(r=0.340,P<0.05),与IFN-γ/IL-4呈负相关趋势(r=-0.332,P<0.05)。结论:SLE患者血清DcR3水平显著升高,可能通过促Th2等作用参与SLE的发生发展。     

碘缺乏和甲状腺功能减退对大鼠仔鼠海马ERK1/2表达的影响

目的观察碘缺乏和甲状腺功能减退对大鼠仔鼠海马细胞外信号调节蛋白激酶1及2(ERK1/2)表达的影响。方法健康2月龄孕Wistar大鼠28只,按体重随机分成对照组、甲状腺功能减退组[按饮水中含丙基硫尿嘧啶(PTU)剂量分为5mg/L组和15mg/L组]和碘缺乏组,每组7只。分别于出生后第7、14、21、28和42天每组随机取5只仔鼠,灌流固定大脑,用组织病理切片和免疫组化染色观察分析海马的ERK1/2表达。结果在出生后14、21、28和42天时,海马CA1和CA3区的ERK1/2表达在PTU5mg/L组、PTU15mg/L组和碘缺乏组显著低于对照组(P0.05)。DG区的ERK1/2表达与对照组相比差异无显著性。出生后7天时,各组间ERK1/2表达差异无显著性。结论碘缺乏和甲状腺功能减退可降低海马CA1和CA3区的ERK1/2表达。     

非肥胖2型糖尿病胰岛素抵抗的病机研究

目的　探讨胰岛素受体数目及其 β亚基自身磷酸化程度改变在 2型糖尿病患者胰岛素抵抗发生中的作用。 方法　 14例非肥胖型 2型糖尿病患者和 12例正常对照者 ,用放射配基结合法测定完整红细胞膜上胰岛素受体数目及其亲和常数 ,按Comi等人所述方法制备红细胞膜蛋白 ,用胰岛素及γ -3 2PATP促发磷酸化反应 ,反应结束后进行不连续垂直板凝胶电泳 ( 0 1%SDS～ 7%5 %PAGE)分离蛋白质 ,并进行放射自显影 ,测定 95KD蛋白质的吸光度。结果　非肥胖型 2型糖尿病患者 ,每个红细胞上胰岛素高亲和力位点数目为 2 1± 13 ,亲和常数为 ( 1 70± 0 93 )× 10 -9LM-1 ,低亲和力位点数目为 95 5± 42 7,亲和常数为 ( 1 44± 0 86)× 10 -7LM-1 ,与正常对照组相比 ,差异无显著性 ;非肥胖型 2型糖尿病患者红细胞膜上 95KD蛋白的磷酸化程度为 8 0 2± 5 60△A/mg蛋白 ,明显低于对照组 2 8 3 8± 15 2 4△A/mg蛋白。结论　胰岛素受体 β亚基自身磷酸化程度的降低可能是非肥胖型 2型糖尿病患者胰岛素抵抗产生的主要因素 ;胰岛素受体数目及其与胰岛素亲和力的改变可能与非肥胖型 2型糖尿病胰岛素抵抗的产生无关     

P13K、P38 MAPK及ERK1/2抑制剂对EGF诱导的血管平滑肌细胞迁移的实验研究

目的 探讨P13K抑制剂Wortmannin、P38 MAPK抑制剂SB202190及ERK1/2抑制剂PD98059对表皮生长因子(EGF)诱导的大鼠主动脉平滑肌细胞(VSMCs)迁移的影响.方法 以1%FCS血清孵育为对照,用EGF(10ng/ml)刺激,平行加入PD98059(PD组)、SB202190(SB组)和Wortmannin(WT),划痕损伤实验分析VSMCs迁移情况.结果 划痕后24 h,EGF组VSMCs迁移较对照组明显(P<0.01),WT组、SB组及PD组与EGF组相比都明显抑制VSMCs迁移(P<0.01),但三个抑制剂组之间差异无统计学意义(P>0.05);划痕后30 h,EGF组VSMCs迁移仍较对照组明显(P<0.01),WT组、SB组及PD组与EGF组相比则明显抑制VSMCs迁移(P<0.01),且三个抑制剂组之间差异有统计学意义,其中SB组较WT组和PD组抑制VSMCs的迁移更明显(P<0.01).结论 EGF诱导VSMCs迁移可能通过P13K、P38 MAPK及ERK1/2途径起作用,且P38 MAPK途径作用更明显.     

P13K、P38 MAPK及ERK1/2抑制剂对EGF诱导的血管平滑肌细胞迁移的实验研究

目的 探讨P13K抑制剂Wortmannin、P38 MAPK抑制剂SB202190及ERK1/2抑制剂PD98059对表皮生长因子(EGF)诱导的大鼠主动脉平滑肌细胞(VSMCs)迁移的影响.方法 以1%FCS血清孵育为对照,用EGF(10ng/ml)刺激,平行加入PD98059(PD组)、SB202190(SB组)和Wortmannin(WT),划痕损伤实验分析VSMCs迁移情况.结果 划痕后24 h,EGF组VSMCs迁移较对照组明显(P<0.01),WT组、SB组及PD组与EGF组相比都明显抑制VSMCs迁移(P<0.01),但三个抑制剂组之间差异无统计学意义(P>0.05);划痕后30 h,EGF组VSMCs迁移仍较对照组明显(P<0.01),WT组、SB组及PD组与EGF组相比则明显抑制VSMCs迁移(P<0.01),且三个抑制剂组之间差异有统计学意义,其中SB组较WT组和PD组抑制VSMCs的迁移更明显(P<0.01).结论 EGF诱导VSMCs迁移可能通过P13K、P38 MAPK及ERK1/2途径起作用,且P38 MAPK途径作用更明显.