低氧预处理通过上调钙网蛋白表达减轻大鼠心肌细胞氧化应激损伤

本文旨在探讨钙网蛋(calreticulin,CRT)是否参与低氧预处理(hypoxic preconditioning,HPC)对心肌细胞氧化应激损伤的保护及其信号转导过程.将原代培养的Sprague.Dawley乳鼠心肌细胞随机分为8组:氧化应激(H2O2)组、短暂低氧(HPC)组、HPC H202组、SB203580(p38 MAPK特异性抑制剂) HPC H2O2组、干扰心肌细胞CRT表达的反义寡核苷酸(antiscnse oligodeoxynucleotides,AS)组、AS H2O2组、AS HPC H202组和对照组,以细胞存活率、乳酸脱氢酶(1actate dehydrogenase,LDH)漏出及流式细胞术检测细胞损伤情况;采用RT-PCR和Western blot分别检测CRT表达和p38MAPK磷酸化水平.结果表明:(1)HPC可减轻氧化应激损伤,与H202组比较,HPC H2O2组细胞存活率增高18.0%,细胞凋亡率和LDH漏出分别降低19.4%和53.0%(均P<0.05);HPC前以SB203580预孵育可消除HPC保护作用,与HPC H202组相比,SB203580 HPC H2O2组细胞凋亡率和LDH漏出分别增高13.1%和96.0%,存活率降低7.3%(均P<0.05);(2)氧化应激明显上调CRT表达(H202组较对照组高7.1倍,P<0.05);HPC也诱导CRT表达上调(HPC组较对照组高2.4倍,P<0.05),但上调程度较H2O2组低59%(P<0.05);即HPC可减轻氧化应激诱导的CRT过表达:(3)AS干扰CRT表达后,HPC保护作用降低,相关性分析显示HPC诱导的CRT适度表达与细胞存活率呈正相关(r=0.8023,P<0.05);(4)HPC前SB203580预孵育可抑制CRT表达上调(分别较HPC H2O2组和HPC组低75%和53%,均P<0.05).上述结果提示,HPC可能通过p38 MAPK信号途径诱导CRT表达上调,减轻心肌细胞氧化应激损伤.     

p38丝裂素活化蛋白激酶介导低氧预处理诱导的内质网应激相关的心肌细胞保护

钙网蛋白（calreticulin,CRT）和caspase-12是重要的内质网（endoplasmic reticulum,ER）应激分子,本实验在心肌细胞低氧／复氧（hypoxia／reoxygenation,H／R）模型上观察低氧预处理（hypoxic preconditioning,HPC）对CRT和caspase-12表达及活化的影响,探讨内质网应激（endoplasmic reticulum stress,ERS）在HPC保护机制中的意义及其细胞信号转导机制。原代培养的Sprague-Dawley乳鼠心肌细胞随机分为6组：H／R组、HPC＋H／R组、SB203580＋HPC＋H／R组、SP600125＋HPC＋H／R组、HPC组和对照组。以细胞存活率、乳酸脱氢酶（lactate dehydrogenase,LDH）活性及流式细胞术检测细胞损伤情况：Western blot方法检测CRT和caspase-12表达、活化及p38丝裂素活化蛋白激酶（mitogen—activated protein kinases,MAPK）、cJun N-terminal kinase（JNK）磷酸化水平。结果表明：（1）HPC具有细胞保护作用,与H／R组比较,HPC＋H／R组细胞凋亡率和LDH漏出分别降低6．6％和70．0％,存活率增高6．4％：HPC前以特异性p38MAPK抑制剂SB203580预孵育消除HPC的保护作用,与HPC＋H／R组相比,细胞凋亡率和LDH漏出分别增高5．4％和2．1倍,存活率降低5．4％,JNK特异性抑制剂SP600125预孵育对HPC的保护作用无明显影响。（2）H／R明显上调CRT表达（较对照组高8．1倍）和caspase-12活性（较对照组高33．2倍）;单独HPC可诱导CRT表达增多（较对照组高2．6倍）,但上调程度较H／R组低60％。H／R前进行HPC降低CRT过表达程度（降低72．4％）及caspase-12活化水平（降低59．6％）。（3）HPC前应用p38MAPK抑制剂,抑制CRT表达上调（分别较HPC＋H／R组和HPC组低63．9％和71．9％）,并消除HPC减轻H／R上调caspase-12活性的作用（较HPC＋H／R组高7．1倍）;HPC前抑制JNK活性对CRT、caspase-12表达和活化均无明显影响。上述结果提示：HPC可激发适当的ERS,抑制H／R诱导的过度ERS,减少ER凋亡信号介导的细胞凋亡。p38MAPK信号途径在HPC诱导的ER应激分子表达、抑制ER凋亡信号分子活化等机制中发挥重要作用。     

短暂低氧／复氧对培养新生兔心肌细胞丝裂素活化蛋白激酶活性的影响

心肌细胞短暂低氧可诱导对后续长时间低氧所致细胞严重损伤的耐力增强,已在心脏预处理（ＰＣ）模型上得到证实,但ＰＣ发生的细胞内信号传导途径目前尚不清楚。我们在培养的新生兔心肌细胞低氧／复氧模型上,观察丝裂素活化蛋白激酶（ＭＡＰＫ）和核蛋白体Ｓ６激酶（Ｓ６Ｋ）活性改变。结果发现：低氧６０ｍｉｎ后、复氧１５ｍｉｎ,细胞总ＭＡＰＫ和核ＭＡＰＫ活性分别较对照组增加９５％和２３０％（Ｐ〈０．０１）;Ｓ６Ｋ活性在     

p38 MAPKα/β和ERK1/2在心肌缺氧预处理信号传递中的不同作用

建立培养乳鼠心肌细胞的缺氧／复氧(A／R)损伤模型和缺氧预处理(APC)模型,以细胞存活率、细胞内超氧化物趋化酶(SOD)活性、丙二醛(MDA)含量、培养上清液乳酸脱氢酶(LDH)活性作为反映心肌细胞损伤的指标。采用细胞外信号调节蛋白激酶(ERK1／2)抑制剂PD98059及丝裂素活化蛋白激酶p38α/β(p38α/β)阻滞剂SB203580干预模型,并以胶内原位磷酸化法测定ERK1／2和p38活性,借以探讨ERK1／2和p38α/β在缺氧预处理保护机制中的作用。结果表明：(1)在APC组,于预处理的缺氧时相给予PD98059,可以完全消除APC的延迟保护作用;在A／R组的缺氧时相加入PD98059对细胞损伤无影响;(2)在APC组的预处理缺氧时相给予p38α／β抑制剂SB203580并不能消除APC的保护作用,而在A／R组的持续缺氧时相给予SB203580则可显著减轻缺氧对细胞的损伤;(3)ERK1／2和p38总活性测定表明,缺氧可激活ERK1／2和p38,它们的活性在缺氧后4h时达到高峰,而经过APC处理后,两者活性高峰提前于缺氧后3h时出现,且峰值显著降低。上述结果提示,预处理过程中ERK1／2的激活可能是缺氧预处理延迟保护机制中细胞信号传递的重要环节,预处理阶段p38α/β的活化不参与APC诱导的延迟保护信号传递过程,p38的过度激活可能是缺氧／复氧损伤过程中的一个致损伤参与因素,而预处理抑制随后持续缺氧阶段p38的过度激活可能是其保护机制的一个环节。     

钙网蛋白在心肌肥大中的研究进展

钙网蛋白（calreticulin,CRT）是内质网中主要的Ca^2＋结合分子伴侣,具有调控细胞Ca^2＋稳态、蛋白质合成与修饰等作用,参与调节细胞凋亡、应激、心血管炎症反应等多种生理和病理生理过程。CRT属于心脏胚胎基因家族,通过调节心肌细胞肌原纤维形成、促进糖原分解、诱导肥大相关基因转录、调节心脏传导系统发育及心肌细胞凋亡等,在心脏发育及心肌肥大的发生、发展过程起重要作用,本文对CRT在心肌肥大中的作用及其信号转导途径予以综述。     

钙网蛋白在心肌肥大中的研究进展

钙网蛋白(calreticulin, CRT)是内质网中主要的Ca2+结合分子伴侣,具有调控细胞Ca2+稳态、蛋白质合成与修饰等作用,参与调节细胞凋亡、应激、心血管炎症反应等多种生理和病理生理过程.CRT属于心脏胚胎基因家族,通过调节心肌细胞肌原纤维形成、促进糖原分解、诱导肥大相关基因转录、调节心脏传导系统发育及心肌细胞凋亡等,在心脏发育及心肌肥大的发生、发展过程起重要作用,本文对CRT在心肌肥大中的作用及其信号转导途径予以综述.     

远端缺血预处理对心肌的保护作用

实验发现,局部缺血预处理对远端(未预处理)心肌组织具有保护作用,而且其它器官的短暂缺血也可实现心肌保护作用,这一现象不同于经典的缺血预处理,因此被命名为远端缺血预处理。研究其机制表明,神经反射调节是机体实现远端缺血预处理保护作用的重要方式,心肌组织中蛋白激酶C激活介导了这一作用。     

重组人钙网蛋白的克隆与原核表达

［摘要］目的: 克隆人钙网蛋白（calreticulin,CRT）并在E.coli中原核表达和纯化。方法：采用RT-PCR 法从人非小细胞肺腺癌A549细胞总RNA中克隆人钙网蛋白cDNA,构建CRT原核表达质粒（pET-15b/CRT）并转化E.coli 的Rossetta菌株。IPTG诱导后,表达蛋白在变性条件下经Ni-NTA 树脂亲和层析纯化,然后透析复性。分别用SDS-PAGE和Western blotting法鉴定CRT表达和纯化状态。结果：从A549细胞总RNA中成功获得人CRT cDNA克隆,重组质粒pET-15b/CRT构建正确。转化pET-15b/CRT的E.coli Rossetta诱导性表达重组人CRT蛋白,该蛋白可经Ni-NTA树脂亲和层析高度纯化。结论：成功建立了CRT原核表达和纯化的实验方法,该方法为后续的CRT蛋白功能研究奠定了基础。     

钙网蛋白及其与肿瘤的关系

钙网蛋白（calreticulin,CRT）是一种普遍存在且高度保守的内质网钙结合蛋白,具有调节钙平衡、协助蛋白质的折叠和加工、参与抗原提呈、血管发生及凋亡的调节等多种生物学功能。最近研究发现,CRT与肿瘤的发生发展、诊断、预后判断以及治疗密切相关。该文介绍钙网蛋白的结构、生物学功能及其与肿瘤的关系。     

Gi／0蛋白介导的信息转导通路在低氧预处理心肌保护中的作用

实验以低氧３ｈ后复氧期间心肌细胞的生存率和ＬＤＨ的释放量为指标,观察Ｇｉ／ｏ蛋白及其下游成分在低氧预处理（ｈｙｐｏｘｉｃ ｐｒｅｃｏｎｄｉｔｉｏｎｉｎｇ,ＨＰ）心肌保护中的作用。与单纯低氧组相比,ＨＰ组（２５ｍｉｎ低氧＋３０ｍｉｎ复氧作为ＨＰ）细胞生存率增高,ＬＤＨ释放减少（Ｐ＜０．０１）。用ＮＥＭ预处理,能完全模拟ＨＰ的心肌细胞保护作用;而用ＰＴＸ阻断Ｇｉ／ｏ蛋白,或Ｆｏｒｓｋｏｌｉｎ和８－Ｂｒ     

脑缺血/缺氧预适应引起的基因、蛋白质表达谱的变化

缺血,缺氧预适应保护作用的分子机制目前尚未充分阐明。最近的研究在大鼠和小鼠脑缺血,缺氧预适应的模型上采用了基因芯片、双向电泳结合质谱分析技术,揭示了大鼠和小鼠脑缺血,缺氧预适应引起的基因和蛋白质表达谱的变化。这些研究发现预适应引起一些新的基因和蛋白质的表达发生改变,深入研究这些基因和蛋白质有可能发现治疗脑中风新的药物靶标。     

Calreticulin induces delayed cardioprotection through mitogen-activated protein kinases

Hypoxic preconditioning (HPC) attenuates tissue injury caused by ischemia/reperfusion. The protective mechanisms of HPC involve up-regulation of the protective proteins and mitigation of cellular calcium overload. Calreticulin (CRT), a Ca(2+)-binding chaperone, plays an important role in regulating calcium homeostasis and folding of proteins. The role of CRT in cardioprotection of HPC and the pathways determining CRT expression during HPC are not clear. In this work, 2-DE and MALDI-MS were employed to analyze CRT differential expression in cardiomyocytes subjected to transient hypoxia. Western blotting analysis was used to detect the CRT expression and activities of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH(2)-terminal kinase (JNK) in myocardium subjected to ischemia with and without HPC and sham operation. The hearts from HPC group were more resistant to sustained ischemia and had much stronger phosphorylation of p38 MAPK, with a reduced phosphorylation of JNK, than controls. The CRT expression was positively correlated with the phosphorylation of p38 MAPK and negatively correlated with the level of JNK phosphorylation. Furthermore, inhibition of the p38 MAPK with SB202190 abolished, while inhibition of the JNK with SP600125 enhanced the CRT up-regulation in cardiomyocytes induced by HPC. The results indicate that HPC up-regulates CRT expression through the MAPK signaling pathways.     

缺血后处理内源性心脏保护的研究进展

再灌注疗法是临床治疗心肌缺血最有效的措施,但会引起再灌注损伤,调动机体内源性保护机制可以减轻再灌注损伤,保护缺血心肌。缺血预处理（ischemic preconditioning,IPC）和后处理（ischemic postconditioning,I-postC）是缺血心脏有效的内源性保护现象,可以减轻缺血再灌注（ischemia／reperfusion,I／R）后心肌坏死与心肌功能障碍,减少恶性心律失常的发生。内源性心脏保护的机制主要是通过诱导触发因子释放,经多条细胞内信号转导途径的介导,作用于多种效应器,影响氧自由基产生、钙超载等I／R损伤的关键环节而发挥心肌细胞保护作用。特别是可以在缺血后实施的I-postC具有良好的临床应用前景。本文以I-postC为重点综述内源性心脏保护作用、机制及其临床应用现状。     

低压低氧延迟预适应小鼠海马差异表达蛋白的分离及鉴定研究

目的:探索低压低氧延迟预适应(HHDP)小鼠海马差异表达蛋白。方法:建立小鼠海马HHDP动物模型后,用含尿素的细胞裂解液提取海马总蛋白质。经等电聚焦、SDS-PAGE电泳、考马斯亮蓝R250染色,比较对照组及HHDP组双向电泳图谱上蛋白质点的差异。切取HHDP组表达增高的蛋白质点,经脱色、胰蛋白酶酶切、提取肽片段,进行MALDI-TOF-MS检测。结果:在正常对照组和HHDP组海马总蛋白双向电泳图谱上,分别检测到481±38和477±21个点,匹配点为169±6个。其中HHDP组比对照组增高大于2倍的有33±10个点,降低大于2倍的有21±12个点。电泳图谱平均相关系数为0.7748±0.0267。有12个点在HHDP组显著增高(P<0.05,n=4),对其进行了肽质量指纹图谱分析,其中8个蛋白点得到相应的肽质量指纹图谱。数据库检索显示其中一个为果糖二磷酸醛缩酶A。3个在蛋白质数据库中没有检索到与之相匹配的任何蛋白,可能为新蛋白。另外4个可找到与其有部分匹配的氨基酸序列,但分子量和等电点与对应蛋白相差悬殊,它们可能具有同源性。结论:小鼠海马HHDP时,果糖二磷酸醛缩酶A等多种蛋白表达发生改变,可能是产生HHDP的重要机制之一。     

DJ‐1 plays an obligatory role in the cardioprotection of delayed hypoxic preconditioning against hypoxia/reoxygenation‐induced oxidative stress through maintaining mitochondrial complex I activity

DJ‐1 was recently reported to mediate the cardioprotection of delayed hypoxic preconditioning (DHP) by suppressing hypoxia/reoxygenation (H/R)‐induced oxidative stress, but its mechanism against H/R‐induced oxidative stress during DHP is not fully elucidated. Here, using the well‐established cellular model of DHP, we again found that DHP significantly improved cell viability and reduced lactate dehydrogenase release with concurrently up‐regulated DJ‐1 protein expression in H9c2 cells subjected to H/R. Importantly, DHP efficiently improved mitochondrial complex I activity following H/R and attenuated H/R‐induced mitochondrial reactive oxygen species (ROS) generation and subsequent oxidative stress, as demonstrated by a much smaller decrease in reduced glutathione/oxidized glutathione ratio and a much smaller increase in intracellular ROS and malondialdehyde contents than that observed for the H/R group. However, the aforementioned effects of DHP were antagonized by DJ‐1 knockdown with short hairpin RNA but mimicked by DJ‐1 overexpression. Intriguingly, pharmacological inhibition of mitochondria complex I with Rotenone attenuated all the protective effects caused by DHP and DJ‐1 overexpression, including maintenance of mitochondria complex I and suppression of mitochondrial ROS generation and subsequent oxidative stress. Taken together, this work revealed that preserving mitochondrial complex I activity and subsequently inhibiting mitochondrial ROS generation could be a novel mechanism by which DJ‐1 mediates the cardioprotection of DHP against H/R‐induced oxidative stress damage.     

模型鼠低氧预适应适宜氧气浓度研究

目的:研究低氧预适应训练的适宜氧气浓度。方法:设计了短期和长期两种间歇性低氧暴露模式,研究了一系列不同浓度的低氧环境对模型鼠体重、血氧饱和度、游泳能力等方面的影响,进而探讨低氧预适应效应与氧气浓度之间的内在联系。结果:模型鼠长期暴露于低氧环境中,其体重增长率逐步下降;在15%～8%的低氧浓度区间,模型鼠血氧饱和度随氧气浓度降低呈现平台似缓慢下降趋势;低氧预适应训练后的模型鼠游泳能力显著提高,经在10%低氧环境中进行低氧预适应训练后的昆明小鼠游泳能力提高最为明显。结论:适当浓度的低氧预适应训练可以改善模型鼠低氧耐受能力,显著提高模型鼠运动能力。15%～10%氧气浓度区间可视为低氧预适应有益作用区间。10%氧气浓度为模型鼠低氧预适应训练的较适宜浓度。     

低氧预适应训练在急进高海拔高原部队中的应用研究

目的：在高海拔高原部队验证低氧预适应降低急性高原反应、改善体能的效果,并观察效果的时间保留规律。方法：拟进驻4300m高原新兵利用低氧呼吸器进行低氧预适应训练,每天上、下午各1h,连续5d,分别于训练结束后第2天和第6天进入高原,观测受试者的急性高原反应症状和体能。结果：低氧预适应训练可显著降低急性高原病的患病率;训练结束后第2天进入高原者VO2 max和PWC170显著降低,而第6天进入高原者,VO2max无明显改变。结论：进驻高海拔高原前进行为期5天的低氧预适应训练可显著降低急性高原病的患病率,训练效果至少可保留5天。     

The timing of propofol administration affects the effectiveness of remote ischemic preconditioning induced cardioprotection in rats

The cardioprotection of remote ischemic preconditioning (RIPC) is abolished under propofol maintained anesthesia. Transient receptor potential vanilloid 1 (TRPV1) channel is present in the heart, and its activation could induce cardioprotection. Therefore, we tested whether the anesthetic propofol administration phase interfered with the RIPC-induced cardioprotection, and RIPC-induced cardioprotection via the cardiac TRPV1 channel. Male Sprague-Dawley rats were subjected to myocardial 30 minutes of ischemia followed by 2 hours of reperfusion. RIPC consisted of three cycles of 5-minute ischemia/reperfusion applied to a hindlimb. Propofol infusion at 12 mg/kg/h was commenced either at 10 minutes before the start of RIPC in the P-pre + RIPC group, or immediately after myocardial ischemia at the onset of reperfusion (P-post + RIPC) while performing RIPC. These two propofol infusion regimes were applied to another two grou bs without RIPC (P-pre and P-post groups). Infarct size (IS) was assessed by triphenyltetrazolium staining. Heart TRPV1 expression was detected by Western blot and immunofluorescence. RIPC significantly reduced myocardial IS compared with the control group (36.7 ± 3% versus 57.2 ± 4%; P < .01). When propofol was started before RIPC, the IS sparing effect of RIPC was completely abolished. However, propofol infusion starting immediately after myocardial ischemia did not affect RIPC-induced cardioprotection. TRPV1 expression significant increase after RIPC, then propofol inhibited the TRPV1 activation of RIPC if given before RIPC but not after. Our results suggest that the timing of propofol administration is critical to preserve the cardioprotection of RIPC. Propofol might cancel RIPC-induced cardioprotection via the cardiac TRPV1 receptor.