【作者】 李慧；

【导师】 张迎梅； 黄德军；

【作者基本信息】 兰州大学 ， 动物学， 2013， 博士

【摘要】 氯酚类化合物常被用作木材防腐剂、杀菌剂、农药和除草剂等,由于其难以降解,在环境中特别是水体中能稳定存在,不仅影响水生生物的生长和繁殖,还可以通过食物链进入人体,危害人体健康,是一类主要的环境污染物。2，4-二氯苯酚(2,4-DCP)作为氯酚类化合物的一种,主要用于除草醚、2,4-滴以及药物硫双二氯酚的合成,由于其大量用于工农业并被释放到环境中,对生物产生毒性影响,美国和中国等许多国家都已将2,4-DCP列入了优先控制污染物的名单。2,4-DCP不仅能影响生物体的生长、发育和生殖,还可以在细胞和分子水平导致氧化应激、促使自由基的产生,影响相关基因的表达,甚至引起细胞死亡。细胞凋亡是指程序性的细胞死亡,可由线粒体信号或死亡受体等介导发生。已有研究证明2,4-DCP可以诱导细胞凋亡,但其诱导细胞凋亡的信号途径和分子机制尚不清楚。本实验以草鱼的原代肝细胞为材料,通过测定2,4-DCP染毒后细胞的活力、细胞凋亡和线粒体膜电位,检测细胞内活性氧(Reactive oxygen species,ROS)的含量及凋亡相关因子caspase-3、Bcl-2相关X蛋白(Bcl-2associated X protein, Bax)、Bcl-2、TNF-α和IAP的mRNA表达情况,探讨了2,4-DCP诱导细胞凋亡的信号途径；通过加入外源性线粒体保护剂乙酰左旋肉碱盐酸(Acetyl-L-carnitine hydrochloride, ALC)进一步验证了线粒体信号途径在2,4-DCP诱导细胞凋亡过程中的作用,最终揭示了2,4-DCP诱导细胞凋亡的具体途径及分子机制,为2,4-DCP毒性作用机理的研究提供一定的科学依据。本论文具体研究结果如下：1.2,4-DCP暴露可以抑制草鱼原代肝细胞的细胞活力。本实验使用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT)检测2,4-DCP对草鱼原代肝细胞细胞活力的影响,结果显示：与对照组相比,2,4-DCP (0.50、1.00mM)暴露12h可以引起草鱼原代肝细胞细胞活力的显著降低(P<0.05)。2.2,4-DCP暴露可以诱导草鱼肝细胞的凋亡。使用荧光显微镜观察和流式细胞仪检测2,4-DCP对草鱼原代肝细胞细胞凋亡的影响,与对照组相比,2,4-DCP (0.10、0.50mM)暴露12h显著增加了草鱼原代肝细胞的凋亡率(P<0.05)。3.2,4-DCP暴露可以引起草鱼肝细胞线粒体膜电位的降低。使用荧光分光光度计检测2,4-DCP对草鱼原代肝细胞线粒体膜电位的影响,检测到与对照组相比,2,4-DCP (0.10、0.50mM)暴露12h可以显著降低草鱼原代肝细胞的线粒体膜电位(P<0.05)。4.2,4-DCP暴露可以导致草鱼肝细胞的ROS的产生。使用流式细胞仪检测细胞内ROS爆发的时间点和ROS水平的变化,与对照组相比,2,4-DCP (0.10、0.50mM)暴露12h没有引起ROS的显著升高(P>0.05),但暴露5h可以引起细胞内ROS的显著升高(P<0.05)。5.2,4-DCP暴露可以诱导草鱼肝细胞凋亡相关基因的改变。使用实时定量PCR (qPCR)检测2,4-DCP对凋亡相关基因mRNA表达量的影响,与对照组相比,2,4-DCP可以导致caspase-3、TNF-α和Bax mRNA的表达量以及Bax/Bcl-2mRNA比值的升高(P<0.05)。6.ALC (5mM)抑制了2,4-DCP导致的草鱼肝细胞的凋亡,并显著恢复了线粒体膜电位(P<0.05),降低了caspase-3、TNF-α和Bax mRNA的表达量(P<0.05)以及Bax/Bcl-2mRNA的比值(P<0.05)。7.2,4-DCP暴露引起草鱼肝细胞IAP mRNA表达量的下降,但对草鱼IAP蛋白的表达没有影响。研究结果显示2,4-DCP导致草鱼肝细胞IAP mRNA表达量的变化。我们进一步利用生物信息学技术手段设计IAP基因的特异性引物,扩增IAP基因片段,运用原核表达系统表达抗原,免疫新西兰大白兔,获得抗血清后,通过亲和纯化得到IAP蛋白特异性的抗体并使用免疫印迹检测了2,4-DCP诱导的草鱼肝细胞中IAP蛋白的表达。然而,与对照组相比,2,4-DCP (0.10、0.50mM)对草鱼肝细胞LAP蛋白的表达没有明显影响(P>0.05)。以上结果表明：2,4-DCP诱导的草鱼肝细胞的凋亡是通过线粒体信号途径介导的。2,4-DCP染毒细胞后通过产生ROS引起氧化应激,降低线粒体膜电位,改变caspase-3和Bax mRNA的表达以及Bax/Bcl-2比值,最终导致细胞凋亡。更多还原

【Abstract】 Chlorophenols (CPs) are widely used as wood preservatives, fungicides, pesticides and herbicides. CPs can exist stably in the environment especially in the water due to its low degradation capacity and are regarded as an important group of environmental contaminants. CPs do not only affect the growth of various aquatic organisms, they can also enter the body through the food chain and endanger human health.2,4-dichlorophenol (2,4-DCP), one of the most abundance of CPs in the environment, is used as solvents, pesticides and pharmaceutical intermediates, has been found to be harmful to humans and animals. Therefore,2,4-DCP has been defined as priority pollutants in USA, China and many other countries.2,4-DCP can affect the growth, development and reproduction of the organism, induce oxidative stress, change the expression of related genes and even lead to apoptosis.Apoptosis, also named programmed cell death, can be mediated by the mitochondrial pathway and the death receptor pathway.2,4-DCP has been demonstrated to induce apoptosis, but the detailed mechanism remains unclear. Therefore, in this study the cell viability, apoptosis, the mitochondrial membrane potential (△Ψm), the mRNA expression of apoptosis-related factors caspase-3, Bcl-2associated X protein (Bax), Bcl-2and TNF-a, the protein expression of LAP and the content of intracellular reactive oxygen species (ROS) in grass carp hepatocytes exposed to2,4-DCP were detected and measured. Exogenous mitochondrial protective agent acetyl-L-carnitine hydrochloride (ALC) was added to further explore the relationship of2,4-DCP-induced apoptosis and mitochondrial pathway. The results were as follows:1.2,4-DCP inhibited the cell viability of grass carp primary hepatocytes.3-(4,5-dimethyl-thiazol-2)-2,5-diphenyl tetrazolium bromide (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) test was used for detecting the cell viability of grass carp hepatocytes.Exposure to2,4-DCP (0.50,1.00mM) for12h significantly reduced the cell viability of grass carp hepatocytes (P<0.05).2.2,4-DCP induced apoptosis in grass carp hepatocytes. Fluorescence microscopy and flow cytometry were used to detect apoptosis of grass carp hepatocytes.Exposure ofgrass carp hepatocytes to2,4-DCP (0.50,1.00mM) for12h significantly increased the apoptosis rate (P<0.05).3.2,4-DCP decreased the△Ψm of grass carp hepatocytes. Fluorescence spectrometry was used to detect the mitochondrial membrane potential of grass carp hepatocytes. The△Ψm was significantly decreased after grass carp hepatocytes being exposed to0.10and0.50mM2,4-DCP for12h(P<0.05).4.2,4-DCP caused ROS production in grass carp hepatocytes. We detected intracellular ROS production in hepatocytes of grass carp by flow cytometry. The results showed that intracellular ROS level was much higher than that in control group when cells were treated with0.10and0.50mM2,4-DCP for5h (P<0.05). However, there were no significant differences (P>0.05) between control and2,4-DCP treatment groups (0.10and0.50mM) after exposure for12h.5.2,4-DCP changed the mRNA expression of apoptosis related genes in grass carp hepatocytes. Real-time quantitative PCR was used to detect the mRNA expression of apoptosis-related gene.0.50mM2,4-DCP increased the mRNA expression of caspase-3, TNF-a, BAX and BAX/Bcl-2mRNA ratio (P<0.05).6. ALC at5mM inhibited the2,4-DCP-induced apoptosis and significantly restored the△Ψm (P<0.05), reduced caspase-3, TNF-a and Bax mRNA expression (P<0.05) and the ratio of Bax/Bcl-2mRNA (P<0.05).7.2,4-DCPdecreased the mRNA expression of IAP, but it had no effect on IAP protein expression in grass carp hepatocytes. We designed a pair of specific primers for IAP gene by bioinformatics technology and amplified DNA fragment.Then we introduced this IAP gene fragment into E.coli to express protein which then was injected into New Zealand white rabbits to prepare antiserum. Finally, the specificanti-IAP polyclonal antibody was obtained by affinity purification. Immunoblot analysis was used to detect the IAP protein expression of grass carp hepatocytes induced by2,4-DCP. The results showed that2,4-DCP (0.10,0.50mM) had no effect on IAP protein expression in grass carp hepatocytes (P>0.05).In conclusion, the present study demonstrated that2,4-DCP-induced apoptosis in primary hepatocytes occurred through the mitochondrial pathway. This process was preceded by the generation of ROS and the disruption of△Ψm, which resulted in the increased mRNA expression of caspase-3and Bax, the elevated Bax/Bcl-2ratio, thus leading to apoptosis. The results reveals the2,4-DCP-induced apoptosis pathway and its molecular mechanism, provides scientific research basis for the research of2,4-DCP.更多还原