【作者】 胡国梁；

【导师】 范利； 何昆仑；

【作者基本信息】 中国人民解放军军医进修学院 ， 老年医学， 2010， 博士

【摘要】 缺血可触发心肌细胞凋亡,如果凋亡持续存在,可导致心肌细胞进行性丢失,甚至发展成为心力衰竭。最近研究表明除了caspase依赖的细胞凋亡途径外,还存在内质网应激诱导的细胞凋亡途径。在缺氧、糖缺乏或药物刺激等应激状态下,细胞内未折叠或错误折叠蛋白积聚,内质网正常功能受损并启动适应性的未折叠蛋白反应。激活的跨膜蛋白PERK使真核翻译起始因子2α亚单位(eIF2a)发生磷酸化,全面抑制蛋白的合成,从而,对细胞起到保护作用。如果应激时间延长,未折叠蛋白反应保护机制不能代偿,最终会通过激活内质网应激凋亡通路蛋白CHOP/GADD153和caspase-12的表达,引起细胞凋亡。衣霉素(tunicamycin, TM)是一种蛋白糖基化抑制剂,可诱导细胞发生凋亡。Salubrinal是一种选择性的磷酸酶抑制剂,可通过抑制eIF2a去磷酸化对抗内质网应激引起的不良反应。但两者在心肌细胞凋亡的诱导和保护作用还未见报道。本实验研究衣霉素是否通过内质网应激凋亡途径引起心肌细胞凋亡,并研究salubrinal对衣霉素诱导的心肌细胞凋亡的保护作用及其信号通路。1.探讨衣霉素在诱导大鼠心肌细胞凋亡中的作用和可能的凋亡通路采用衣霉素对乳鼠原代心肌细胞进行诱导,通过MTT、DNA梯带、流式细胞等方法观察TM与心肌细胞凋亡之间的关系。用real time PCR和Western blot法检测凋亡过程中GRP78、eIF2α、P-eIF2a和caspase-12表达的变化。结果发现：①衣霉素能诱导心肌细胞发生凋亡,并且这种凋亡作用呈浓度依赖性和时间依赖性；②衣霉素早期可引起内质网应激未折叠蛋白反应分子伴侣P-eIF2α和GRP78 mRNA及蛋白表达水平的升高,在后期引起内质网应激凋亡通路蛋白CHOP/GADD153上调和caspase-12裂解,提示衣霉素引起的内质网应激,从未折叠蛋白反应转向前凋亡反应。2.探讨salubrinal对衣霉素诱导的乳鼠原代心肌细胞凋亡的保护作用及机制采用MTT法、流式细胞、TUNEL的方法检测salubrinal对TM诱导的心肌细胞凋亡的保护作用,用高内涵筛选、Hoechst染色和Western印迹法检测凋亡过程中GRP78.eIF2α、[.P-eIF2a.CHOP/GADD153和裂解的caspase-12表达的变化。结果发现：①salubrinal在100μM浓度范围内对心肌细胞是无毒安全的；②salubrinal可引起心肌细胞P-eIF2α蛋白表达水平的升高,且这种升高表现为浓度依赖性；③salubrinal本身不引起心肌细胞内质网应激,并对衣霉素诱导的心肌细胞凋亡起到了保护作用,且这种保护作用呈浓度依赖性；④salubrinal对衣霉素诱导的心肌细胞凋亡有拮抗作用,可能通过诱导eIF2α磷酸化和抑制CHOP和cleaved caspase一12等内质网应激通路蛋白表达来实现。更多还原

【Abstract】 Ischemia can trigger the apoptosis of cardiomyocyte, if the condition persists, progressive loss of cardiomyocyte were ensued, and even develop into heart failure. Recent studies have shown that in addition to caspase dependent apoptotic pathway, there also exists the endoplasmic reticulum stress-induced apoptotic pathway. Under cellular stress conditions, such as hypoxia,glucose deprivation and drugs, the accumulation of unfolded/misfolded proteins, disrupts the proper function of ER and initiates the unfolded protein response (UPR) to cope with this adverse situation. Activated PERK phosphorylates eukaryotic translation initiation factor 2 subunit a (eIF2a), which inhibited global protein synthesis and leads to protection of cells,However, prolonged ER stress with the failure of cellular protective mechanisms by UPR will eventually activate the expression of CHOP/GADD153 and caspase-12, two molecular chaperones in ERS apoptosis pathway, and finally results in cell apoptosis.Tunicamycin (tunicamycin, TM), an inhibitor of glycosylation of proteins, can induce cellular apoptosis. Salubrinal was identified as a selective inhibitor of phosphatases, through inhibiting the dephosphorylation of eIF2a, which result in a halt of protein synthesis and permit cells to recover from endoplasmic reticulum (ER) stress. But in terms of cardiomyocyte apoptosis induced by TM and protective effect by salubrinal has not been reported. Here, we examined whether TM initiates the apoptosis of cardiomyocyte through activating the death pathway of ER stress, and investigated the role and signaling pathways,which salubrinal act on the apoptosis of myocardiomyocyte induced by tunicamycin.1. To investigate tunicamycin in the induction of cardiomyocyte apoptosis and the possible pathway of apoptosis.To investigate the relationship between tunicamycin and cardiomyocyte apoptosis, neonatal rat cardiomyocyte treated by tunicamycin was detected by MTT assay,DNA Ladder and flow cytommetry. Real time PCR and western blot methods were used to observe the expression of GRP78, eIF2a, phosphorylation of eukaryotic initiation factor 2-a(P-eIF2a) and caspase-12. The results showed that:①Tunicamycin can induce cardiomyocyte apoptosis in a concentration-dependent and time-dependent manner;②Unfolded protein response (UPR),a cytoprotective response, was detected by the induction of GRP78, and P-eIF2a in the primary stage, laterlly the ER stress response switched from UPR to a pro-apoptotic response as demonstrated by the upregulation of CHOP/GADD153 and processing of caspase-12.2. To investigate the protective effects and related mechanisms of salubrinal on neonatal rat cardiomyocyte apoptosis induced by tunicamycinMTT, flow cytometry, TUNEL were used to detect the protective effects of salubrinal on cardiomyocyte apoptosis induced by tunicamycin,and the expressions of GRP78, eIF2a, P-eIF2a, CHOP/GADD153 and cleaved caspase-12 during the apoptosis were detected by high content screening, Hoechst staining and Western blot. We found that①Within the range of 100μM, salubrinal was non-toxic to cardiomyocyte;②The expression of P-eIF2a protein in cardiomyocyte treated by salubrinal was increased in a concentration-dependent manner.③Salubrinal does not cause endoplasmic reticulum stress by itself, and plays protective roles in the cardiomyocyte apoptosis induced by tunicamycin in a concentration-dependent manner;④The protective effect of salubrinal on tunicamycin-induced cardiomyocyte apoptosis, may through inhibiting the dephosphorylation of eIF2a and donwn-regulating the expressions of CHOP/GADD153 and processing of caspase-12.更多还原