【作者】 徐菲菲；

【导师】 刘秀华；

【作者基本信息】 中国人民解放军军医进修学院 ， 病理学与病理生理学， 2009， 博士

【摘要】 缺血性心脏病严重危害人类健康,尽早恢复血流是减轻缺血心肌损伤的关键,溶栓、经皮穿刺冠状动脉腔内血管成型术和冠状动脉旁路手术等再灌注疗法是临床治疗心肌缺血最有效的措施。但是再灌注疗法受缺血组织血管再通时间的限制并存在再灌注损伤等问题。阐明缺血/再灌注（ischemia/reperfusion,I/R）损伤的发病机制是医学界亟待解决的重大问题。内质网（endoplasmic reticulum,ER）作为真核细胞中调控蛋白质折叠、Ca²⁺稳态和应激反应最重要的细胞器之一,对应激刺激非常敏感,缺血缺氧、葡萄糖/营养物质匮乏、ATP耗竭、大量自由基的产生及Ca²⁺稳态破坏等均可引起内质网功能障碍,触发内质网应激（ER stress,ERS）。一定程度的ERS可促进内质网功能恢复,长时间严重的ERS则破坏细胞Ca²⁺稳态、诱导ERS相关性细胞死亡（ER associated death,ERAD）,造成组织损伤,是I/R损伤的机制之一。内质网应激主要表现为葡萄糖调节蛋白类（glucose-regulated proteins,GRPs）、钙网蛋白（calreticulin,CRT）、蛋白质折叠酶等物质表达上调和caspase-12、CHOP/GADD153等促凋亡因子的表达及活化。其中钙网蛋白（CRT）是内质网（ER）中主要的Ca²⁺结合分子伴侣,通过调节ER Ca²⁺贮存而影响细胞浆游离Ca²⁺水平,并作为ER分子伴侣参与蛋白质折叠,具有调节Ca²⁺稳态、蛋白质折叠、细胞凋亡等多种生物学功能,在缺血/再灌注损伤发病中的作用引起高度关注。本室前期工作证实缺血/再灌注明显上调CRT,缺血预处理通过适度上调CRT发挥内源性心肌细胞保护作用,文献报道CRT在缺血/再灌注中具有双重作用,阐明CRT在缺血/再灌注中的作用及其机制具有十分重要的意义。本工作采用乳大鼠心肌细胞缺氧/复氧（hypoxia/reoxygenation,H/R）模型模拟在体心肌I/R损伤,首先证实心肌细胞H/R损伤触发严重ERS,引起心肌细胞凋亡;给予ERS诱导剂衣霉素（tunicamycin,TM）可模拟H/R损伤;在此基础上通过转染pCDB-CRT质粒诱导或siRNA抑制内质网应激分子CRT表达,研究CRT的表达变化对H/R损伤的影响,证实CRT是引起内质网应激相关凋亡的关键分子;进一步探讨CRT介导ERS相关凋亡的信号途径,并研究缺氧后处理（hypoxic postconditioning,H-postC）对于CRT介导内质网应激相关凋亡的影响。主要方法与结果如下:一、H/R触发严重ERS,引起心肌细胞损伤原代培养的Sprague-Dawley乳大鼠心肌细胞缺氧4h复氧24 h复制H/R模型,并以不同浓度ERS诱导剂衣霉素（tunicamycin,TM）直接诱导心肌细胞内质网应激,采用台盼蓝排斥实验和乳酸脱氢酶（lactate dehydrogenase,LDH）活性测试盒分别检测细胞存活率和LDH漏出情况,采用Annexin V-FITC细胞凋亡检测试剂盒检测细胞凋亡率,采用内质网特异性荧光染料Dapoxyl观察内质网形态变化;并采用RT-PCR和/或Western blot检测内质网应激分子（PERK、GRP78、CRT、CHOP等）及凋亡相关分子（caspase-12、caspase-3、Bax和Bcl-2等）的表达变化,证实H/R与TM同样引起CRT、CHOP、caspase-12、caspase-3、Bax等表达上调,PERK磷酸化增强,并出现明显的心肌细胞凋亡,提示H/R触发严重ERS,引起心肌细胞凋亡。二、CRT是H/R损伤过程中介导ERS相关凋亡的关键分子本部分工作分别在TM诱导内质网应激和心肌细胞H/R模型上,证实转染pCDB-CRT质粒诱导CRT过表达可加重内质网应激反应,活化ERS相关凋亡途经、加重心肌细胞损伤;siRNA干扰CRT表达可减轻上述细胞损伤,抑制ERS相关凋亡途经,提示CRT是H/R损伤过程中介导ERS相关凋亡的关键分子。三、CRT介导H/R损伤的信号通路CRT外源性过表达时,RT-PCR检测结果显示CHOP mRNA上调;Western blot检测证实PERK磷酸化及CHOP蛋白表达增加,ERS相关凋亡途径活化;CRT表达抑制时,PERK磷酸化降低,CHOP mRNA及蛋白表达均下调,ERS相关凋亡途径受抑制;相关分析显示PERK磷酸化及CHOP表达量与CRT正相关,提示CRT可能通过促进PERK磷酸化,上调CHOP表达介导ERS相关凋亡。检测内质网Ca²⁺-ATP酶SERCA(sarco（endo）plasmic reticulumCa²⁺-ATPase)活性、胞浆游离Ca²⁺浓度[Ca²⁺]。和钙调神经磷酸酶（calcineurin,CaN）活性,相关分析显示CRT表达量与SERCA活性负相关,而与[Ca²⁺]_i、CaN活性正相关,提示CRT可能通过抑制SERCA活性,降低内质网Ca²⁺重摄取能力,使[Ca²⁺]_i升高,促进CaN活化,引起心肌细胞损伤。四、缺氧后处理抑制CRT介导的ERS相关凋亡减轻H/R损伤H-postC抑制H/R诱导的CRT过表达,使PERK磷酸化程度降低,CHOP转录及蛋白表达下调,抑制ERS相关凋亡途经活化,上调SERCA活性、下调[Ca²⁺]_i与CaN活性,减轻心肌细胞损伤;外源性过表达CRT可削弱H-postC的保护作用,表现为PERK磷酸化增加,CHOP表达上调,ERS相关凋亡途经激活,提示H-postC的保护机制与抑制CRT介导的ERS相关凋亡有关。结论:CRT过表达参与心肌细胞H/R损伤,其机制一方面通过PERK磷酸化上调CHOP表达,促进ERS相关凋亡途径活化;另一方面可能与抑制SERCA活性,升高[Ca²⁺]_i,激活CaN有关。内源性保护现象H-postC可减轻CRT介导的H/R损伤。更多还原

【Abstract】 Ischemic heart diseases seriously endanger human health. Restoration of blood flow as soon as possible is the key to reduce ischemic myocardial injury. Reperfusion therapies such as thrombolysis, percutaneous transluminal coronary angioplasty and coronary artery bypass surgery, are the most effective treatments to salvage myocardium following ischemic injury. However, the effect of reperfusion therapy is restricted by vascular recanalization time and reperfusion injury. To interpret the pathogenesis of ischemia/reperfusion injury is the major issue need to be resolved urgently in the medical field.Endoplasmic reticulum （ER） is the most important organelle in eukaryotic cell, adjusting protein folding, Ca²⁺ homeostasis and stress response. ER is very sensitive to stress stimulation. Ischemia/hypoxia, glucose/nutrient deprivation, ATP depletion, a large number of free radicals production or disturbance of Ca²⁺ homeostasis can cause dysfunction of endoplasmic reticulum and trigger endoplasmic reticulum stress. A certain degree of ERS improves the restoration of ER function, but excessive and prolonged ERS, as one of the mechanisms of ischemia/reperfusion injury, destroy Ca²⁺ homeostasis and induce ER associated cell death, and result in tissue injury. ERS induces upregulation of ER molecular chaperones such as glucose-regulated proteins（GRPs）, calreticulin（CRT）, folding enzymes, and activation of pro-apoptotic factors such as caspase-12 and CHOP/GADD153. Among these factors, calreticulin（CRT） is an essential Ca²⁺-binding /storage chaperone resident protein of endoplasmic reticulum or sarcoplasmic reticulum found across a diverse range of species. The protein is involved in the regulation of intracellular Ca²⁺ homeostasis and endoplasmic reticulum Ca²⁺ storage capacity, and is also an important molecular chaperone involved in "quality control" within secretory pathways. CRT plays an important role in apoptosis, cell adhesion, gene expression and autoimmunity. It is focus on the effect of calreticulin in the pathogenesis of ischemia/reperfusion injury. Our previous work has confirmed that ischemia/reperfusion obviously upregulate CRT expression and ischemia preconditioning protects cardiomyocyte through moderately upregulating CRT expression. It is reported that CRT has a dual role in I/R injury. It is very important to investigate the effect and molecular mechanism of CRT in I/R injury.The present study used neonatal rat cardiomyocyte hypoxia/reoxygenation model to simulate in vivo cardiac ischemia/reperfusion injury. At first, we confirmed that H/R triggered severer ERS and induced cardiomyocyte apoptosis. ERS inducer tunicamycin（TM） simulated H/R injury. In order to investigate whether CRT is the key factor to induce ERS-related apoptosis in H/R injury and its signaling pathway, pCDB-CRT plasmid transfection or siRNA interference were used to interfere with CRT expression. Then the effect of hypoxic postconditioning（H-postC） on CRT-mediated H/R injury was investigated. Major methods and results were as follows:1. H/R triggers severer ERS and induces cardiomyocyte apoptosis.Neonatal rat cardiomyocytes were prepared from Sprague-Dawley rats aged24 hours. H/R was induced by 4 hours’ hypoxia（H） followed by 24 hours’ reoxygenation（R）. Different concentrations of TM were used to induce ERS directly. Morphological studies, lactate dehydrogenase （LDH） leakage, and flow cytometry were employed to assess cell apoptosis and necrosis. ER specific resident fluorochrome Dapoxyl was used to observe ER morphological changes. RT-PCR and/or Western blot were used to detect expression of ERS molecules such as ATF4, p-PERK, GRP78, CRT, CHOP and apoptosis-related molecules such as caspase-12, caspase-3, Bax and Bcl-2. It was suggested that H/R as the same as Tm up-regulate expression of CRT, CHOP, caspase-12, caspase-3 and Bax, and increase phosphorylation of PERK, and induce cardiomyocyte apoptosis.2. CRT is the key factor to induce ERS-related apoptosis in H/R injuryIn the modols of TM-induced ERS and cardiomyocyte H/R, we showed that pCDB-CRT plasmid transfection aggratated ERS, activated ERS-related apoptosis pathway and led to excessive cell injury, while siRNA interference attanuated cell injury and inhibited the ERS-related apoptosis pathway.3. Signalling pathway of CRT-mediated H/R injuryCRT overexpression upregulated the expression of CHOP both in mRNA （by RT-PCR） and protein （by Western blot）, and the phosphorylation of PERK. Correlation analysis indicated that PERK phosphorylation and CHOP expression was positive con-elation with CRT expression. Activities of sarco（endo）plasmic reticulum Ca²⁺-ATPase and calcineurin, and intracellular Ca²⁺ concentration ([Ca²⁺]i) were dectected, which showed a negative correlation between CRT expression and SERCA activity and a positive correlation between CRT expression with [Ca²⁺]i and CaN activity.4. H-postC suppresses CRT-mediated ERS-related apoptosis induced by H/RH-postC suppressed the up-regulation of CRT, CHOP, and phosphorylation of PERK and inhibited ERS-related apoptosis. H-postC also up-regulated SERCA activity, down-regulated [Ca²⁺]i and CaN activity, and attenuated cell injury. Extraneous over-expression of CRT decreased the cardioprotective effect of H-postC, suggesting that suppression of CRT-mediated ERS-related apoptosis was invlolved in cardioprotection of H-postC.Conclusion: CRT overexpression mediates H/R injury im cardiomyocytes through the pathways as follws: （1） CRT overexpression induces ERS-related apoptosis through phosphorylating PERK and up-regulating CHOP expression. （2） CRT overexpression down-regulates SERCA activity, up-regulates [Ca²⁺]i and CaN activity. H-postC attenuates CRT-mediated ERS-related apoptosis during H/R injury.更多还原