【作者】 李兰；

【导师】 王子仁；

【作者基本信息】 兰州大学 ， 动物学， 2010， 博士

【摘要】 有机磷化合物(organophosphorus compounds, OPCs)在全世界范围内被广泛应用于工业、农业和医学领域中。由于其广泛的应用,OPCs也是分布广泛的环境污染物,对包括人类在内的多种生物具有很强的毒性。因此,迄今为止,OPCs的毒性效应和毒理学机制一直是众多科学研究者关注的对象,并进行了许多这方面的体外和在体研究。近年来,越来越多的体外和在体的研究结果表明诱导凋亡是OPCs的一种新发现的毒理学机制,并且凋亡性的细胞死亡在OPCs诱导的损伤效应中起重要作用。因此,很有必要阐明OPCs诱导的凋亡的分子机制,目前已经有一些科学家进行了这方面的研究。目前已有的实验结果表明,钙信号与凋亡的诱导和调节有密切的关系,内质网通路是目前为止发现的三条主要的凋亡信号通路之一。但是尚未有实验研究内质网通路是否参与了OPCs诱导的凋亡,以及钙信号是否在OPCs诱导的凋亡早期的信号传递中发挥作用。本研究以对氧磷(paraoxon, POX)诱导的小鼠T淋巴细胞瘤细胞系EL4细胞凋亡为模型,研究钙信号是否在对氧磷诱导的EL4细胞凋亡早期的信号传递中发挥作用,以及内质网通路是否参与了该凋亡的过程。在本研究中我们采用实时监测激光扫描共聚焦显微技术(laser scanning confocal m icroscopy, LSCM)检测对氧磷诱导的EL4细胞凋亡早期(0-2 h)细胞内钙信号的变化,并采用荧光显微技术检测对氧磷诱导的EL4细胞凋亡的形态学变化和凋亡率的变化。Caspase-12是内质网通路中的一个重要信号分子。在本研究中我们采用免疫组织化学方法(immunohistochemistry, IHC)检测对氧磷诱导的EL4细胞凋亡过程中caspase-12表达的变化。本研究中EGTA被用于螯合细胞外的钙离子,肝素(heparin)和普鲁卡因(procaine)分别被用来抑制内质网上的IP3受体相关的钙通道和兰尼定(ryanodine)受体相关的钙通道,从而抑制钙离子从内质网的释放。本研究的实验结果表明,对氧磷在浓度范围1-10 nM时处理EL4细胞16 h后,能以浓度依赖的模式诱导细胞凋亡率显著上升。1-10 nM对氧磷还以浓度依赖的模式诱导EL4细胞内钙信号在凋亡的早期(0-2 h)增强。分别用EGTA.肝素和普鲁卡因对EL4细胞进行预处理可以部分地抑制对氧磷诱导的EL4细胞内钙信号增强和细胞凋亡。此外,对氧磷还可以以浓度依赖的模式上调caspase-12的表达量。分别用EGTA、肝素和普鲁卡因对EL4细胞进行预处理能显著抑制对氧磷诱导的caspase-12表达量上升现象。这些实验结果显示,对氧磷可以在EL4细胞中诱导钙信号的上升,此种上升发生在对氧磷诱导的EL4细胞凋亡过程的早期(0-2 h)。钙信号的上升来源于细胞外钙离子的内流和内质网存储的钙离子的释放,内质网存储的钙离子主要是通过IP3受体相关的钙通道和兰尼定受体相关的钙通道释放。本研究首次证明钙信号是对氧磷诱导的EL4细胞凋亡过程中重要的上游信号,内质网通路也参与了该凋亡过程的信号传递。更多还原

【Abstract】 Organophosphorus compounds (OPCs) are frequently utilized in agriculture, industry and medicine all over the world. Because of their wide uses, OPCs are also widespread environmental pollutants and potent toxicants to many kinds of organisms, including human beings. Therefore, until the present time, toxic effects and toxicological mechanisms of OPCs have been attended to and been studied in vitro or in vivo by many researchers. In recent years, more and more studies have indicated that induction of apoptosis is a new toxic effect of OPCs, in vitro or in vivo, and that apoptotic cell death may play an important role in OPC-mediated impairing effects. It is necessary to elucidate molecular mechanisms involved in OPC-induced apoptosis, and some researchers have investigated signaling pathways in apoptosis induced by OPCs.According to results obtained now, intracellular calcium signaling has been strongly implicated in induction of apoptosis and regulation of the apoptotic signaling pathways and the endoplasmic reticulum (ER)-associated pathway is one of the three major apoptotic pathways which have been found to date. Until the present time, no one has investigated whether intracellular calcium signals were involved in OPC-induced apoptosis at the early stage of apoptotic process, and whether the ER-associated pathway participated in apoptosis induced by OPCs. In the present study, we used the experimental model in which paraoxon (POX) induced apoptosis in murine EL4 T-lymphocytic leukemia cells. The objective of this study is to investigate whether intracellular calcium signals were involved in POX-induced apoptosis in EL4 cells at the early stage of apoptotic process, and whether the ER-associated pathway participated in this apoptotic process.We used real-time laser scanning confocal microscopy (LSCM) to examine POX-induced changes of intracellular calcium signals at the early stage (0-2 h) of POX-induced apoptosis in EL4 cells, and we investigated apoptotic rates and morphological changes of EL4 cells after treatment with POX using fluorescent microscopy. Caspase-12 is one of key elements in the ER-pathway. We also evaluated changes of caspase-12 expression in POX-induced apoptosis in EL4 cells, using immunohistochemistry (IHC). In the present research, EGTA was used to chelate Ca2+in extracellular medium; heparin and procaine were used as the specific antagonists to IP3 receptor (IP3 R)-associated Ca2+channels and ryanodine receptor (Ry R)-associated Ca2+channels respectively, to inhibit Ca2+efflux from the ER.POX (1-10 nM) increased intracellular calcium signals of EL4 cells in a dose-dependent manner at the early stage (0-2 h) of POX-induced apoptosis, and apoptotic rates of EL4 cells after treatment with POX for 16h also increased in a dose-dependent manner. Pre-incubation with EGTA, heparin or procaine could partly inhibit POX-induced intracellular calcium elevation and apoptosis in EL4 cells. Additionally, POX could up-regulate caspase-12 expression in a dose-dependent manner, and pre-incubation with EGTA, heparin or procaine could significantly inhibit POX-induced increase of caspase-12 expression.Our results suggest that POX induced intracellular calcium increases in EL4 cells at the early stage (0-2 h) of POX-induced apoptotic process, which included the mobilization of Ca2+stored in the ER and Ca2+influx from extracellular medium. Ca2+release from the ER was via IP3 R-associated Ca2+channels and Ry R-associated Ca2+channels. The present study firstly proved that calcium signaling was an important upstream messenger in POX-induced apoptotic process in EL4 cell, and the ER-associated pathway was also involved in this apoptosis.更多还原