【作者】 董建军；

【导师】 李小鹰； 鲁晓春；

【作者基本信息】 中国人民解放军军医进修学院 ， 老年医学， 2009， 博士

【摘要】 缺血性心脏病是目前心力衰竭发生的主要原因之一,其发病率和死亡率呈逐年上升的趋势。随着细胞生物学和分子生物学的飞速进展,人们对缺血性心脏病发生、发展机制有了进一步认识,其防治措施也取得了较大进展。基因治疗作为一种新的治疗策略为缺血性心脏病的治疗提供了新的途径。近年来有关内质网应激（endoplasmic reticulum stress,ERS）的信号转导通路与效应的研究非常活跃。研究发现,ERS介导的细胞凋亡在心肌缺血、心力衰竭进展过程中起重要作用。有研究表明,主动脉缩窄引起的心脏压力过负荷可以诱导ERS,导致心肌细胞凋亡增多,而转基因高表达GRP78、GRP94等内质网分子伴侣可减轻甚至取消内质网应激、促进细胞存活。钙稳态失衡是诱导ERS发生的重要原因。在参与Ca²⁺循环的各种Ca²⁺调节蛋白中,肌浆网钙ATP酶2a（sarcoplasmic reticulum calcium ATPase 2a,SERCA2a）的蛋白水平和活化程度是细胞内Ca²⁺稳态的决定性因素。SERCA2a表达降低和活化程度下降是心力衰竭心肌舒缩功能异常的关键环节。既往研究表明,转基因过表达SERCA2a可以提高心脏的泵血功能,改善血流动力学状况。最近有研究证实,过表达SERCA2a还可以延缓心肌梗死后心力衰竭的发生,但具体机制尚未阐明。基于以往研究及现有文献报道,我们推测过表达SERCA2a可能有助于减轻心肌缺血缺氧造成的内质网应激,具有保护心肌细胞的作用。本研究在离体细胞水平,观察缺氧情况下心肌细胞内质网应激的主要过程。以重组病毒rAd-SERCA2a为载体,观察过表达SERCA2a对缺氧诱导心肌细胞内质网应激及相关凋亡途径的影响。旨在为过表达SERCA2a的基因治疗方法应用于缺血性心力衰竭提供理论基础。首先,本研究利用人胚胎肾细胞（HEK293细胞）扩增重组腺病毒rAd-SERCA2a及rAd-GFP,并通过Touchdown-PCR鉴定,证实重组入腺病毒基因的正确性。利用携带报告基因的重组腺病毒rAd-GFP转染心肌细胞,以及利用乳酸脱氢酶（LDH）观察转染对心肌细胞损伤程度,确定最佳的MOI值。为开展基因靶向性转染心肌细胞打下基础。其次,将体外培养的心肌细胞进行缺氧处理,观察不同时间内心肌细胞发生内质网应激的情况。采用Western blot方法检测内质网应激标志性蛋白GRP78、CHOP、Caspase-12表达变化,DNA ladder及流式细胞术检测心肌细胞凋亡情况。结果显示,心肌细胞缺氧6h后GRP78蛋白表达水平逐渐升高,至缺氧24h达峰值,随后表达有所下降。说明缺氧能够诱导内质网分子伴侣GRP78表达升高,但其表达水平具有一定的时效性。即缺氧早期（24h内）,GRP78蛋白表达丰度逐渐增加,其对细胞的保护作用也逐渐增强,而持续长时间缺氧（24h以上）,GRP78表达相对下降,对细胞的保护作用减弱。缺氧12h后凋亡因子CHOP和Caspase-12蛋白表达逐渐增加,36h后表达水平显著升高。流式细胞术检测显示,随着缺氧时间的延长,心肌细胞凋亡率逐渐上升。提示缺氧诱导心肌细胞发生内质网应激,并且随着缺氧时间延长,内质网应激介导的细胞保护机制转向细胞损伤机制,心肌细胞最终发生凋亡。随后,以重组腺病毒载体过表达SERCA2a后,再给予缺氧或衣霉素刺激,观察过表达SERCA2a对内质网应激及细胞凋亡的影响。结果显示,与缺氧组比较,SERCA2a+缺氧组GRP78表达无明显变化,但是CHOP蛋白表达水平明显下降,而Caspase-3蛋白表达也较缺氧组降低39.7%。SERCA2a+缺氧组的细胞凋亡率较缺氧组明显降低（P＜0.05）。提示过表达SERCA2a蛋白可减轻缺氧诱导心肌细胞内质网应激的强度,对内质网应激介导的心肌细胞损伤具有重要的防护作用。其作用机制:过表达SERCA2a虽然不能提高内质网应激分子伴侣GRP78等保护性蛋白的表达水平,但是可以抑制凋亡因子CHOP、Caspase-3等表达,进而减轻心肌细胞凋亡。最后,利用Fura-3和激光共聚焦显微镜观察细胞内游离Ca²⁺的变化。结果显示,缺氧12h后心肌细胞胞浆内游离钙浓度显著增高,提示细胞内钙超载。过表达SERCA2a+缺氧组细胞内荧光强度较缺氧组显著降低,差异有统计学意义（P＜0.05）。提示,过表达SERCA2a可增加肌浆网对Ca²⁺的摄取能力,有利于缓解心肌细胞缺氧时胞浆内钙浓度的持续升高,从而抑制钙超载诱导的细胞凋亡。更多还原

【Abstract】 Ischemic heart disease(IDH) is one of the leading contributors to the heart failure currently and the morbidity and mortality of IDH are increasing greatly. With the rapid development of in the field of cell biology and molecular biology, the mechanism of IDH was further elucidated and the effective prevention measures were discovered.As one of strategies for the treatment of IDH,Gen therapy is a new method.In recently years,the signaling and effect of endoplasmic reticulum stress(ERS) is extensively investigated.The apoptosis mediated by ERS play an important role in the progression of myocardial ischemia and heart failure.The heart’s overloaded pressure by coarctation of aorta could induce ERS,and the more apoptosis of cardiac myocytes were discovered in myocardium as a consequence.The overexpression of molecular chaperones of endoplasmic reticulum such as glucose regulated protein 78(GRP78) and GRP94 could attenuate the level of ERS response,then more myocytes undergoing all kinds of stimuli survived.The homeostasis of calcium is one of very important inducers for ERS.In the regulatory proteins involving the cycling of calcium,the level and activity of sarcoplasmic reticulum calcium ATPase 2a(SERCA2a) plays a key role for the homeostasis of calcium.The reduced level and activity of SERCA2a is the to critical to the normal contraction and relaxation of heart.Previous studies showed that overexpression of SERCA2a by transgene technique improved the pumping function of heart and hemodynamics.Recent research identified that overexpression of SERCA2a delayed the occurrence of heart failure induced by cardio infarction,but the exact mechanism was not clarified until now.Here we supposed that overexpression of SERCA2a attenuate the effect of ERS induced by myocardial ischemia and protect the cardiac myocytes from stressors.We observed the process of ERS under the ischemic conditions and the influence of SERCA2a overexpression on ERS and the associated apoptosis in cardiomyocytes. Firstly,HEK293 cells were used to the amplification of recombinant adenovirus and the target gene was identified by polymerase chain reaction(PCR). The rAd-GFP vector and LDH was utilized to determine the appropriate multiplicity of infection.Secondly,cultured myocytes were treated under hypoxia.The protein immunoblotting was used to measure the change of expression of Glucose Regulated Protein 78 and CHOP and Caspase-12.The techniques of DNA ladder and flow cytometry were occupied to examine the apoptosis of cardiomyocytes. The results showed that the expression of GRP78 protein increased in 6 hours and approached the highest level in 24 hours in hypoxia.A decreased trend of GRP78 expression was observed after 24 hours.Those observations indicated that hypoxia could induce the time-limited expression of GRP78 in cardiomyocytes. With the prolonged hypoxia,the protein of CHOP and Caspase-12 expressed increasingly and the cell’s apoptosis rate was higher and higher.We conclude that hypoxia could induce ERS in cardiomyocytes.The protective effect of ERS could not counteract the insulted effect with prolonged hypoxia.Finally,the apoptosis of cardiomyocytes was observed.Thirdly,although the level of GRP78 did not change significantly,CHOP and Caspase-3 decreased by 52.7%and 39.7%respectively in SERCA2a+hypoxia group compared with the hypoxia group.The apoptosis rate of cardiomyocytes decreased by 66%compared with the hypoxia group.Those results indicated that overexpression SERCA2a could attenuate the level of ERS induced by hypoxia. The role of overexpression of SERCA2a can protect the cardiomyocytes from stimulus such as hypoxia.Finally,the level of cellular free calcium was measured by Fluo-3 and confocal microscopy.The elevated calcium concentration was observed in 12 hours under hypoxia conditions.The overload of calcium in cardiac myocytes was decreased with overexpression of SERCA2a.Those results indicated that overexpression SERCA2a could augment the ability of endoplasmic reticulum to uptake calcium and the apoptosis was depressed.更多还原