凋亡诱导因子介导缺氧/复氧致肥大心肌细胞凋亡的作用

心肌细胞凋亡导致心肌组织合胞体功能丧失,最终使代偿性心肌肥大向心力衰竭转化。过去的研究已经确认天门冬氨酸特异性半胱氨酸蛋白酶(caspartate-specificcysteinylproteinase,caspase)依赖机制在心肌细胞凋亡中的作用,但对caspase非依赖机制即凋亡诱导因子(apoptosis-inducingfactor,AIF)在心肌细胞凋亡中的作用尚不明确。本研究应用血管紧张素Ⅱ(0.1μmol/L培养12h)诱导培养的小鼠肥大心肌细胞,利用三气孵箱建立缺氧/复氧模型以模拟缺血再灌注。应用RT-PCR、Westernblot、siRNA基因转染、Hoechst33258染色法检测AIF在mRNA和蛋白质水平的表达及细胞凋亡的变化,分析AIF在缺氧/复氧致肥大心肌细胞凋亡中的意义。结果如下:(1)与对照组比较,缺氧8h组(H8h)和缺氧12h组(H12h)AIFmRNA及蛋白表达水平均显著升高(mRNA:0.52±0.04及0.85±0.10vs0.29±0.08,P<0.05;蛋白质:2.07±0.15和3.12±0.19vs0.29±0.04,P<0.05),即随缺血时间的延长,AIFmRNA及蛋白表达水平均显著增加。(2)与对应单纯缺氧组比较,缺氧后给予复氧刺激,H8h/R组和H12h/R组AIFmRNA及蛋白表达水平均显著升高(mRNA:1.09±0.12和1.41±0.23,P<0.05;蛋白质:4.57±0.25和5.71±0.27,P<0.05)。仅在H8h/R及H12h/R组,可见AIF核转位显著增加。(3)AIFsiRNA转染可显著抑制肥大心肌细胞AIF的表达,对缺氧时细胞凋亡无明显影响(P>0.05),但可显著降低缺氧/复氧诱导的肥大心肌细胞凋亡率(P<0.05)。同时抑制AIF及caspase-3活性,可显著加强单一抑制剂对缺氧/复氧诱导的肥大心肌细胞凋亡的抑制作用。(4)抑制caspase-3活性对缺氧/复氧诱导的AIF核转位无明显影响。上述结果提示,缺氧/复氧时AIFmRNA、蛋白表达和核转位均显著增加,且在缺氧/复氧诱导肥大心肌细胞凋亡中具有重要的作用。     

乌司他丁对大鼠脑缺血再灌注损伤后的保护作用及其机制研究

目的:探究乌司他丁在脑缺血再灌注损伤中的脑保护作用机制。方法:原代分离培养雄性SD大鼠脑皮质细胞,部分细胞经siRNA沉默HSP70基因。细胞先以无糖培养基在低氧条件下培养,12 h后复糖复氧模拟体外缺血再灌注损伤,并实施乌司他丁预处理干预,流式细胞术检测各组细胞的凋亡率,western-blotting检测Bcl-2,Bax,HSP70,JNK和p-JNK蛋白的表达。结果:与对照组比较,模型组脑组织细胞凋亡率明显增多(P0.05)、Bcl-2和Bax的表达量均有上调,Bcl-2/Bax的比值显著降低(P0.01)、HSP70的表达无显著变化;与模型组比较,乌司他丁处理组脑组织细胞凋亡率明显降低(P0.05)、Bax的表达量显著下调(P0.05),Bcl-2/Bax的比值显著上调(P0.05),HSP70的表达显著上调(P0.05),JNK的表达无显著变化、p-JNK则显著下调(P0.05)。HSP70沉默后乌司他丁的脑保护作用消失,对以上蛋白的表达无显著影响。结论:乌司他丁可能是通过上调HSP70表达进而抑制JNK信号转导通路对缺血再灌注引起的脑损伤起保护作用。     

缺氧复氧心肌细胞中HSP90对AKT表达的调控作用

研究新生大鼠缺氧复氧心肌细胞中热休克蛋白90(HSP90)的表达对AKT表达的影响,探讨HSP90在PI3K/AKT信号通路中的作用。方法:建立新生大鼠心肌细胞缺氧复氧模型,通过运用HSP90阻断剂Geldanamycin(GA),分另以MTT法检测心肌细胞的活力、透射电镜观察心肌细胞超微结构改变、Western印迹法分析大鼠心肌细胞中HSP90表达变化与总AKT蛋白的相关性。结果:缺氧复氧心肌细胞中HSP90和AKT表达量均有明显升高,阻断HSP90后,AKT表达量明显下降,此时心肌细胞活力明显下降,细胞超微结构受损明显。结论:AKT对缺氧复氧引起的心肌细胞损伤有内源性保护作用,此作用的发挥与HSP90和AKT的结合密切相关,HSP90对缺氧复氧中的心肌细胞AKT表达有显著影响。     

汉坦病毒包膜糖蛋白G2重组腺病毒的构建及其在Hela细胞中的表达

本研究旨在构建可表达汉坦病毒(HTNV)糖蛋白G2的重组腺病毒。应用PCR方法扩增G2编码基因,经T/A克隆、测序鉴定后再亚克隆到腺病毒shuttle载体pAd5-CMV中并用磷酸钙沉淀法分别将携带G2编码基因的重组腺病毒shuttle载体与携带报告基因eGFP的腺病毒骨架质粒共转染HEK293细胞,包装、扩增、纯化后得到携带HTNV糖蛋白G2编码基因的重组腺病毒;用重组腺病毒感染Hela细胞并收获蛋白,间接免疫荧光、Western blotting检测蛋白表达。经酶切鉴定表明已成功构建了携带G2基因的重组腺病毒载体;RT-PCR鉴定表明目的基因能够在感染重组腺病毒的Hela细胞中转录;荧光显微镜观察重组腺病毒感染的Hela细胞,可见报告基因eGFP的表达;间接免疫荧光法和Western blotting均证实表达产物可被抗G2单克隆抗体所识别,表明糖蛋白G2在感染细胞中得到了表达。本研究成功构建了可表达HTNV包膜糖蛋白G2的重组腺病毒,转染宿主细胞可稳定表达目的蛋白,为HTNV糖蛋白G2的结晶、结构解析研究以及新型汉坦病毒疫苗的研制奠定了基础。     

抑制或激活泛素-蛋白酶体途径对大鼠肺泡巨噬细胞内质网应激的影响

目的:研究肺泡巨噬细胞(NR8383)不同蛋白酶体激活程度对内质网应激的影响。方法:构建UbG76V-GFP融合蛋白,将含有UbG76V-GFP的质粒导入NR8383细胞,筛选出可稳定表达UbG76V-GFP的细胞系,通过蛋白酶体抑制剂(MG132)、蛋白酶体激活剂(阿霉素)干预蛋白酶体活性。荧光显微镜观察不同蛋白酶体活性下大鼠肺泡巨噬细胞在缺氧复氧2 h、4 h、6 h时蛋白酶体活性,Western blot及PCR技术检测不同蛋白酶体活性下大鼠肺泡巨噬细胞在缺氧复氧2 h、4 h、6 h时泛素化蛋白及内质网应激相关基因的表达。结果:在缺氧复氧2 h、4 h、6 h这3个时间点,加入MG132组大鼠肺泡巨噬细胞绿色荧光及泛素化蛋白(Ubiquitin)表达明显降低(P0.05),而PCR及Western blot示内质网应激基因BIP(免疫球蛋白结合蛋白)、XBP-1(X-盒结合蛋白)和CHOP(C/EBP同源蛋白)平均扩增量及蛋白表达量明显增加(P0.05);加入阿霉素组大鼠肺泡巨噬细胞在缺氧复氧2 h、4h、6 h表现出相反的实验结果,绿色荧光及Ubiquitin蛋白相对表达均明显增加(P0.05),而PCR及Western blot示内质网应激基因BIP、XBP-1和CHOP平均扩增量及蛋白表达量明显增加(P0.05)。结论:本实验结果表明活细胞泛素-蛋白酶体活性程度与内质网应激存在紧密联系,外源性增强泛素蛋白酶体活性会抑制内质网应激,外源性减弱泛素蛋白酶体活性会增强内质网应激。     

Hsp70 对低氧大鼠海马DG 区神经细胞p53、p-p38 表达的研究

目的:探讨热休克蛋白70(HSP70)高表达对低氧大鼠海马DG区神经细胞p53、p-p38表达的影响。方法:将大鼠海马DG区神经细胞在41℃温浴1h诱导HSP70高表达,然后低氧培养,Western-blot检测细胞HSP70、p53、p-p38的表达。结果:温浴可以诱导HSP70高表达;HSP70高表达可抑制p53和p-p38表达。结论:HSP70保护低氧诱导的细胞凋亡可能与抑制p53和p-p38表达有关。     

STAT3小干扰RNA的构建及对缺氧复氧人肾小管上皮细胞凋亡的影响

目的:构建信号转导与转录因子3(STAT3)小干扰RNA(siRNA)表达载体,并观察其对缺氧复氧后人肾小管上皮细胞(HKC)凋亡的影响。方法:设计3对人STAT3 siRNA靶序列,用DNA重组技术克隆至质粒pRNAT-U6.1/neo中,构建重组质粒pRNAT-U6.1-STAT3 siRNA,检测并筛选出最佳抑制效率的siRNA质粒载体。重组质粒转染至缺氧复氧后HKC细胞,Western blotting和Real Time-PCR测定STAT3蛋白和mRNA表达量,流式细胞仪测定细胞凋亡,间接荧光法测定Bcl-2和Bax表达的变化。结果:靶向STAT3基因表达的质粒载体构建成功,并筛选出抑制效率最佳的重组质粒。缺氧复氧后HKC细胞STAT3表达、凋亡率和Bax/Bcl-2比值增加;缺氧复氧后HKC细胞转染重组质粒后STAT3表达、凋亡率和Bax/Bcl-2比值明显降低。结论:成功构建并筛选最佳抑制效率的靶向STAT3的重组质粒载体。该载体可有效抑制缺氧复氧后HKC细胞中STAT3信号转导通路的活化,并进一步通过上调Bcl-2、下调Bax蛋白的表达,从而抑制细胞凋亡。     

缺氧复氧诱导脐静脉内皮细胞凋亡的机制

目的探讨缺氧复氧诱导人脐静脉内皮细胞凋亡发生的机制.方法体外培养人脐静脉内皮细胞,随机分为5组:缺氧0h(对照组)、3h、6h、12h、24h复氧组.向培养瓶内通入95%N2和5%CO2按不同时间孵育,随后通入5%CO2和95%空气复氧2h,建立内皮细胞缺氧复氧模型.采用台盼蓝染色、TUNEL技术对凋亡和死亡细胞进行定量分析,DNA电泳观察内皮细胞凋亡的形态学.Western blot检测细胞凋亡调节蛋白Bcl-2和Bax表达强度,同时检测丝裂原活化蛋白激酶(MAPK)中磷酸化ERK1/2的表达.采用凝胶成像分析系统灰度扫描检测蛋白质表达相对量.结果缺氧复氧后内皮细胞凋亡明显,而且内皮细胞凋亡数随缺氧时间延长而增多(P<0.05).Western blot表明缺氧复氧增强内皮细胞Bax的表达,对Bcl-2的表达量和磷酸化ERK1/2没有明显影响,使Bcl-2/Bax比值减小.结论缺氧复氧可以诱导内皮细胞凋亡,证实缺氧复氧上调促凋亡蛋白Bax的表达,对抑制凋亡蛋白Bcl-2的表达无显著影响.首次证实缺氧复氧诱导内皮细胞凋亡是取决于Bcl-2/Bax比值,而不是通过MAPK磷酸化途径.     

HSP70 高表达保护低氧诱导的大鼠海马DG 区神经细胞凋亡

目的:研究诱导HSP70高表达对低氧引起的大鼠海马DG区神经细胞凋亡的保护作用。方法:大鼠海马DG区神经细胞分别在41℃温浴2h和加入砷酸钠诱导HSP70高表达。对照低氧而不预热,并用HSP70反义寡核苷酸链抑制HSP70合成,观察HSP70与低氧大鼠海马DG区神经细胞凋亡的关系。DNA碎片法检测细胞凋亡,Western Blotting检测HSP70。结果:预热和砷酸钠都可以诱导细胞HSP70高表达;HSP70高表达可以明显减少低氧诱导的细胞凋亡。在预热前导入HSP70反义核酸,可以降低HSP70抑制细胞凋亡的作用。结论:HSP70高表达可以保护细胞由于低氧引起的细胞凋亡。     

腺病毒介导的人抑瘤素M基因对A375人黑色素瘤细胞的抑制作用

目的:构建抑瘤素M(OSM)重组腺病毒载体,研究其对人黑色素瘤细胞A375的抑制作用。方法:以PEGZ-OSM重组质粒为模板,通过PCR技术扩增出OSM片段,采用腺病毒载体的基因重组和体外包装技术获得表达与人OSM氨基酸序列相同的重组腺病毒子Ad-OSM,感染A375细胞,用荧光显微镜、RT-PCR、Western blot法检测OSM在A375细胞中的转录和表达;荧光显微镜观察A375细胞的形态学改变;MTT法和流式细胞术(FCM)检测Ad-OSM对A375细胞的生长抑制和细胞周期的抑制效应;半定量RT-PCR法检测OSM基因表达对A375细胞中的Bax、Bcl-2基因表达的影响。结果:基因测序和PCR分析结果显示,成功构建了Ad-OSM腺病毒表达载体;RT-PCR和Western blot法检测到OSM基因在A375细胞中的转录和表达;OSM基因的表达对A375细胞增殖有明显抑制作用,并可诱导细胞凋亡,OSM基因可通过上调细胞中Bax和下调Bcl-2基因表达诱导细胞凋亡。结论:成功构建了Ad-OSM腺病毒表达载体,感染OSM基因可明显抑制A375人黑色素瘤细胞的生长,诱导其凋亡,该现象可能是通过改变Bax、Bcl-2基因表达水平来发挥抗肿瘤作用。     

Hsp70 regulates the doxorubicin-mediated heart failure in Hsp70-transgenic mice

The aim of this study was to investigate the potential protective effect of the Hsp70 protein in the cardiac dysfunction induced by doxorubicin (DOX) and the mechanisms of its action. For this purpose, we used both wild-type mice (F1/F1) and Hsp70-transgenic mice (Tg/Tg) overexpressing human HSP70. Both types were subjected to chronic DOX administration (3 mg/kg intraperitoneally every week for 10 weeks, with an interval from weeks 4 to 6). Primary cell cultures isolated from embryos of these mice were also studied. During DOX administration, the mortality rate as well as weight reduction were lower in Tg/Tg compared to F1/F1 mice (P < 0.05). In vivo cardiac function assessment by transthoracic echocardiography showed that the reduction in left ventricular systolic function observed after DOX administration was lower in Tg/Tg mice (P < 0.05). The study in primary embryonic cell lines showed that the apoptosis after incubation with DOX was reduced in cells overexpressing Hsp70 (Tg/Tg), while the apoptotic pathway that was activated by DOX administration involved activated protein factors such as p53, Bax, caspase-9, caspase-3, and PARP-1. In myocardial protein extracts from identical mice with DOX-induced heart failure, the particular activated apoptotic pathway was confirmed, while the presence of Hsp70 appeared to inhibit the apoptotic pathway upstream of the p53 activation. Our results, in this DOX-induced heart failure model, indicate that Hsp70 overexpression in Tg/Tg transgenic mice provides protection from myocardial damage via an Hsp70-block in p53 activation, thus reducing the subsequent apoptotic mechanism.     

Differential inhibition of Hsc70 activities by two Hsc70-binding peptides

The ability of two high-affinity Hsc70-binding peptides [FYQLALT (peptide-Phi) and NIVRKKK (peptide-K)] to differentially inhibit Hsc70-dependent processes in rabbit reticulocyte lysate (RRL) was examined. Both peptide-Phi and peptide-K inhibited chaperone-dependent renaturation of luciferase in RRL. Peptide-Phi, but not peptide-K, blocked Hsp90/Hsc70-dependent transformation of the heme-regulated eIF2 alpha kinase (HRI) into an active, heme-regulatable kinase. In contrast, peptide-K, but not peptide-Phi, inhibited Hsc70-mediated suppression of the activation of mature-transformed HRI. Furthermore, HDJ2 (Human DnaJ homologue 2), but not HDJ1, potentiated the ability of Hsc70 to suppress the activation of HRI in RRL. Mechanistically, peptide-K inhibited, while peptide-Phi enhanced, HDJ2-induced stimulation of Hsc70 ATPase activity in vitro. The data presented support the hypotheses that peptide-Phi acts to inhibit Hsc70 function by binding to the hydrophobic peptide-binding cleft of Hsc70, while peptide-K acts through binding to a site that modulates the interaction of Hsc70 with DnaJ homologues. Overall, the data indicate that peptide-Phi and peptide-K have differential effects on Hsc70 functions under quasi-physiological conditions in RRL, and suggest that therapeutically valuable peptide mimetics can be designed to inhibit specific functions of Hsc70.     

The neuroprotective potential of heat shock protein 70 (HSP70)

In response to many metabolic disturbances and injuries, including stroke, neurodegenerative disease, epilepsy and trauma, the cell mounts a stress response with induction of a variety of proteins, most notably the 70-kDa heat shock protein (HSP70). Whether stress proteins are neuroprotective has been hotly debated, as these proteins might be merely an epiphenomenon unrelated to cell survival. Only recently, with the availability of transgenic animals and gene transfer, has it become possible to overexpress the gene encoding HSP70 to test directly the hypothesis that stress proteins protect cells from injury. A few groups have now shown that overproduction of HSP70 leads to protection in several different models of nervous system injury. This review will cover these studies, along with the potential mechanisms by which HSP70 might mediate cellular protection.     

Hsp70 in oncology

Hsp70 classes of molecular chaperones are highly conserved in all organisms and play an essential role in the maintenance of cellular homeostasis. Hsp70s assist nascent chain protein folding and denatured proteins, as well as the import of proteins to the organelles, and solubilization of aggregated proteins. ATPase function is required for Hsp70 function. Hsp70s use ATP hydrolysis driven mechanism for substrate protein binding and release. Various Hsps are unregulated in cancers but their significance for tumor growth is poorly understood. Studies have linked Hsp70 to several types of carcinoma. Human Hsp70s allow proliferation of cancer cells and suppress apoptotic and senescence pathways. This review presents Hsp70s role for growth of transformed cells and the current state of Hsp70 as a drug target along with recent patents in humans in this particular area.     

Otto Appel 70 Jahre

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