内源性一氧化碳对肺动脉平滑肌细胞增殖基因表达谱的影响

目的 运用芯片技术研究内源性一氧化碳（CO）刺激下血管平滑肌细胞内增殖相关基凼表达谮的变化。方法 取SD大鼠肺动脉平滑肌细胞进行离体培养,用血小板源性生长因子（PDGF,20ng／m1）和Hemin（20μmol／L）进行刺激后,用Affymetrix表达基因谱芯片检测其基因表达谱变化。结果 Hemin刺激与PDGF刺激相比差异表达基因366条,与增殖相关的差异表达基因21条,上调11条,下调10条。其中原来在PDGF刺激下上调表达的一些基因（Map2k3、cyclinD1、PDGFA、mybL1、a．nape10、MMP14等）在经内源性CO刺激后其表达则显著下调。结论 内源性CO很可能是通过作用于MAPK途径来抑制PDGF的促增殖功能,同时伴有cyclinD1、cyclinG1、P21等的参与。     

脾酪氨酸激酶对大鼠肺血管平滑肌细胞增殖的作用

目的：研究脾酪氨酸激酶（spleen tyrosine kinase, Syk）在血小板源性生长因子BB（platelet-derived growth factor-BB, PDGF-BB）诱导大鼠肺血管平滑肌细胞（PVSMCs）增殖中的作用。方法：体外培养雄性Sprague-Dawley大鼠PVSMCs,经鉴定后选用3~5代细胞,经PDGF-BB诱导细胞增殖后,用3种不同剂量Syk选择性抑制剂piceatannol干预,以MTT比色法测定细胞增殖,3H-TdR掺入法检测细胞DNA合成,流式细胞仪检测细胞周期,荧光定量PCR、Western blot分别检测细胞Syk mRNA和蛋白表达水平。结果：PDGF-BB刺激后,Syk蛋白表达水平明显增高,细胞显著增殖;经Syk抑制剂piceatannol干预后,随着Syk mRNA和蛋白表达水平的降低,细胞的增殖受到明显抑制,其抑制作用与剂量大小具有相关性。结论：在PDGF-BB诱导下的大鼠肺血管平滑肌细胞增殖中,Syk可能起促进作用。 ［中国当代儿科杂志,2010,12（11）：886-890］     

血红素加氧酶 -1抗人血管内皮细胞凋亡的实验研究

目的 转染pcDNA3HO1入人血管内皮细胞（HUVEC）并表达,研究血红素加氧酶-1（HO-1）保护细胞凋亡的作用。方法 将构建好的质粒用DOTAP包裹转入细胞中表达：用CCLR破碎细胞后,SDS-PAGE鉴定表达量,采用TNF-α诱导细胞凋亡,以流式细胞仪测定细胞的凋亡率;采用Hemin和SnPP,分别刺激细胞,促进和抑制HO-l在细胞中的表达。结果 经Hemin处理后细胞的凋亡率均在20％以下,而SnPP处理后细胞的凋亡率大幅上升,最高可达到95％以上。实验数据显示HO-1基因表达被抑制时细胞凋亡率是诱导时的5～20倍。结论 细胞凋亡率与质粒转染效率和HO-l表达量均成反比,提示HO-l对细胞有保护作用,可以抑制细胞凋亡,存临床具有广泛的应用前景。     

内源性一氧化碳对肺动脉平滑肌细胞凋亡基因表达谱的影响

目的：运用芯片技术研究内源性一氧化碳(CO)刺激下血管平滑肌细胞凋亡相关基因表达谱的变化。方法：取Sprague-Dawley (SD)大鼠肺动脉平滑肌细胞进行离体培养,用血小板源性生长因子BB(platelet-derived growth factor,PDGF-BB, 20 ng/mL)和高氯血红素(hemin, 20 μg/mL）进行刺激后2 h,用Affymetrix表达基因谱芯片检测其基因表达谱变化。结果：PDGF刺激后,Map2k3(P38)信号通路中相关差异表达基因Kras、Nras、丝分裂原蛋白激酶的激酶Map2k3 、细胞周期素CyclinD1、CyclinH、CyclinL1等的表达显著上调,而周期素依赖性蛋白激酶抑制因子P27的表达显著下调。Hemin刺激后,P53依赖途径中的一些基因如:Gadd45α、P21、Trp53inp1表达显著上调。结论内源性CO所引发的血管平滑肌细胞的凋亡可能与P53调控途径密切相关。［中国当代儿科杂志,2010,12(11)：882-885］     

血红素加氧酶-1抗人血管内皮细胞凋亡的实验研究

目的转染pcDNA3HO1入人血管内皮细胞(HUVEC)并表达,研究血红素加氧酶_1(HO_1)保护细胞凋亡的作用。方法将构建好的质粒用DOTAP包裹转入细胞中表达。用CCLR破碎细胞后,SDS_PAGE鉴定表达量,采用TNF_α诱导细胞凋亡,以流式细胞仪测定细胞的凋亡率;采用Hemin和SnPP分别刺激细胞,促进和抑制HO_1在细胞中的表达。结果经Hemin处理后细胞的凋亡率均在20%以下,而SnPP处理后细胞的凋亡率大幅上升,最高可达到95%以上。实验数据显示HO_1基因表达被抑制时细胞凋亡率是诱导时的5~20倍。结论细胞凋亡率与质粒转染效率和HO_1表达量均成反比,提示HO_1对细胞有保护作用,可以抑制细胞凋亡,在临床具有广泛的应用前景。     

福辛普利对大鼠肾小球系膜细胞增殖及分泌细胞外基质的影响

目的观察福辛普利（FOS）对脂多糖（LPS）诱导的大鼠肾小球系膜细胞（GMC）增殖及分泌细胞外基质（ECM）的影响。方法建立体外培养的大鼠GMC,鉴定后3—10代用于实验。实验分为五组：对照组、LPS刺激组（LPS组）、福辛普利（FOS）高、中、低剂量组（分别为FOS1组、FOS2组、FOS3组）。甲基噻唑基四唑（MTT）比色法测定24h和48h两个时间点各组细胞增殖,酶联免疫吸附试验（ELISA）测定6h、12h和24h细胞培养上清液中ECM蛋白含量;荧光半定量RT-PCR法检测ECM纤维连接蛋白（LN）β2mRNA表达的变化。结果（1）LPS可诱导GMC增殖,FOS可抑制LPS诱导的GMC增殖;（2）正常培养的大鼠GMC可分泌一定量的ECM,LPS组在各时间点分泌ECM均高于对照组（P〈0．01）,而FOS各组分泌ECM均低于LPS组（P〈0．01）;（3）正常培养的大鼠GMC可表达一定量LNβ2 mRNA,LPS组在各时间点LNβ2mRNA表达量均高于对照组,FOS各组表达量均低于LPS组。结论LPS可诱导GMC增殖且分泌ECM增加,FOS可抑制LPS诱导的GMC增殖,从蛋白和mRNA两个水平抑制LPS诱导的GMC分泌ECM增加,为FOS对肾脏的保护作用提供了理论依据。     

转录因子GATA-3与支气管哮喘的治疗

转录因子GATA-3是具有锌指结构的GATA家族中的一员,可诱导辅助性T细胞(Th)2分化、调控Th1/Th2平衡及调节一些细胞因子的表达,且嗜酸性细胞在受抗原刺激后也表达GATA-3,表明GATA-3在支气管哮喘的发病中起重要作用。GATA-3反义寡核苷酸、GATA-3抑制剂、GATA-3-decoy寡核苷酸可抑制GATA-3表达,为从分子水平治疗支气管哮喘提供了一个崭新的切入点。     

去铁胺诱导HL—60细胞凋亡及对c—myc基因表达的影响

目的 探讨去铁胺（DFO）对HL－60细胞凋亡的诱导作用及对c-myc基因表达的影响。方法 HL－60细胞与不同浓度的去铁胺培养6、12、24、48h。分别测定细胞活力,观察形态学变化,用流式细胞学检测和DNA凝胶电泳等观察细胞凋亡及免疫组化方法检测c-myc基因表达等指标。结果 DFO可降低HL－60细胞活力、抑制HL－60细胞增殖、诱导其凋亡,并使c-myc基因表达增加,其作用随培养时间延长及DFO浓度增加而明显。结论 DFO可影响HL－60细胞DNA的合成,诱导其凋亡,c-myc的表达增加可能是铁剥夺诱导HL－60细胞凋亡的机制之一。DFO可作为治疗或辅助治疗白血病的药物用于临床,将有重要的临床实用价值。     

RNAi阻断NPM-ALK基因表达及对大细胞间变性淋巴瘤细胞的影响(英文)

目的：应用RNA干扰技术抑制大细胞间变性淋巴瘤细胞系(Karpas299)中NPMALK融合基因表达,观察其对肿瘤细胞生长的影响。方法：针对NPMALK融合位点设计两个siRNA序列siRNAI与siRNAII,经PCR反应构建含U6启动子siRNA正义和反义线性表达载体,通过脂质体转染Karpas299细胞,应用实时荧光定量RTPCR、Westernblot检测siRNA片段对NPMALKmRNA和蛋白表达的抑制作用,MTT、Hoechst荧光染色检测siRNA对肿瘤细胞生长的影响。结果：siRNAI可导致NPMALKmRNA下降约75%(P<0.05),转染72h后可导致蛋白表达下降;转染siRNAII细胞NPMALKmRNA下降为35%(P<0.05),但蛋白水平无明显改变。转染siRNAI的细胞可抑制Karpas299细胞的增殖和诱导凋亡发生,siRNAII则无明显的抑制增殖和诱导凋亡作用。结论：含有针对NPMALK融合位点特异siRNA序列的U6表达载体,可特异地抑制NPMALK基因mRNA和蛋白的表达,并能抑制大细胞间变性淋巴瘤肿瘤细胞株Karpas299细胞的增殖,导致肿瘤细胞凋亡增加,提示NPMALK融合基因的异常表达与大细胞间变性淋巴瘤形成密切相关,为研究NPMALK基因功能和大细胞间变性淋巴瘤基因靶向治疗提供了新策略。     

survivin基因在地塞米松诱导CEM细胞凋亡过程中的变化(英文)

目的：糖皮质激素诱导白血病细胞凋亡的确切机制尚不清楚。存活素(survivin)是凋亡抑制蛋白(inhibitorsofapoptosisprotein,IAPs)家族的成员,与凋亡抑制、肿瘤细胞增殖、血管形成及耐药正相关。该研究探讨地塞米松诱导急性淋巴白血病细胞系CEM凋亡过程中survivin基因的表达。方法：将在体外培养的对数生长期的CEM细胞浓度调至2×105个/mL,接种于24孔培养板中,用终浓度分别为0.1,0.5,1,5,10μM地塞米松处理,以不加任何药物的CEM细胞作为对照组,培养后24,48,72h取样。台盼蓝拒染法测定细胞活力,流式细胞仪解析地塞米松诱导CEM细胞凋亡,WesternBlot、RT-PCR方法分别检测survivin蛋白和基因的表达。结果：0.1,0.5,1,5,10μM地塞米松于48h开始明显抑制CEM细胞的生长,抑制效果呈时间、剂量依赖方式。随着地塞米松剂量增大,凋亡细胞比例逐渐增加。5μM地塞米松处理12~48h,凋亡细胞比例从14.9%升至46.2%。5μM地塞米松处理降低了survivin蛋白表达,12h降至对照组的54.6%,24h降至45.5%,48h降至15.8%,72h降至9.7%。survivinmRNA表达在5μM地塞米松处理后也被下调,处理后6h降至对照组的76.4%,12h降至67.3%,24h降至55.0%,36h降至49.9%,48h降至38.3%,72h降至18.3%。结论：地塞米松诱导CEM细胞凋亡与下调survivin基因表达有关。     

Update on Cochrane data on paediatric respiratory diseases

INTRODUCTION: Systematic reviews seek to describe and summarise the best evidence for a given intervention by pooling data from relevant quality clinical trials. The Cochrane Collaboration has fostered the development and dissemination of systematic reviews throughout the world. We have identified and summarised The Cochrane systematic reviews of relevance to the paediatric pulmonologist. METHODS: We performed an expert search of the Cochrane Database of Systematic Reviews using a combination of medical subject headings and free text terms relating to paediatric respiratory disease. RESULTS: The search identified 120 systematic reviews with interventions specific to children with some relevance to pulmonary disease, and 327 reviews with interventions relating to pulmonary disease in adults and children. After pragmatic exclusions, 81 reviews were sorted by disease and 59 of these are discussed in detail. CONCLUSIONS: There are now many systematic reviews that make a positive contribution to paediatric pulmonology. The majority of reviews (69%) found evidence that either confirmed or refuted an accepted practice. The remaining reviews concluded that the evidence for an accepted practice is poor and sometimes wholly absent. Clinicians must be aware that lack of evidence of effect is not the same as evidence of lack of effect. Caution must be exercised before applying the conclusions of systematic reviews based upon adult data to childhood disease.     

Passive smoking by asthmatics: its greater effect on boys than on girls and on older than on younger children

In 415 nonsmoking asthmatic children who were seen consecutively, asthma symptoms were more severe if the mother was a smoker than if she was a nonsmoker. This applied to both sexes but was more marked in boys than in girls. There were also other indications that sons were the more severely affected: the forced expiratory volume at 1 second, the forced expiratory flow rate during the middle half of the forced vital capacity, and the provocation concentration of histamine needed to result in a 20% decrease in the forced expiratory volume at 1 second were significantly decreased only in the sons, and lung function test results were significantly less in sons than in daughters of mothers who smoked. When the 415 children were stratified according to age, lung function improved significantly with increasing age in the children of nonsmokers; in children of smokers, by contrast, symptoms and lung function test results became progressively worse. As well, there was a correlation between these indications of asthma severity and the number of years the child had been exposed to the mother's smoke. It appeared that, compared with girls, boys were more sensitive to passive smoking, and that its adverse effect increased with age and with duration of exposure.