【作者】 徐进；

【导师】 徐立红；

【作者基本信息】 浙江大学 ， 劳动卫生与环境卫生学， 2008， 博士

【摘要】 铅是一种常见的重金属,由于其延展性好、抗腐蚀能力强、熔点低等特性,在工业生产和日常生活中被广泛应用。铅无法被生物所降解,所以其在环境各相和生物系统中持续存在,造成了全球性的空气、水、土壤铅污染的问题,并对人类健康构成持续性的威胁。铅暴露能引起人体的生理、生化及行为等方面发生异常。大量的研究已证实铅毒性可涉及到人体全身,累及神经、血液、心血管、泌尿、生殖、骨骼、内分泌及免疫等系统;其中神经毒性由于对学龄前儿童的严重危害而受到更为广泛的关注。近些年来,铅的毒性机理也得到了较为充分的研究,发现铅主要通过与蛋白结合和模拟钙的功能产生毒性。最近许多研究还发现铅能引起大鼠脑、睾丸、成纤维、肺和视网膜细胞发生凋亡,提示细胞凋亡可能在铅毒性中起了重要的作用。细胞凋亡是一种高度受调控的细胞死亡方式,是应对外界刺激,维持机体平衡的重要手段。细胞凋亡的调控十分复杂,主要有三条信号途径参与其中:死亡受体途径、线粒体途径和内质网途径。凋亡的信号途径涉及了数百个基因和蛋白,从而构成了一个复杂的网络系统,对细胞的生或死进行精密的调控。以往的研究中提示,线粒体途径可能在铅诱导细胞凋亡中起了主要作用。然而,铅是如何导致线粒体功能失调以及哪些上游信号参与了线粒体途径尚未明确。此外,铅能扰乱内质网钙的稳态,而内质网途径是否也参与了铅诱导细胞凋亡的过程尚无研究报道。综上所述,本研究以神经元模式细胞—PC 12细胞为研究对象,验证乙酸铅能否诱导PC 12细胞发生凋亡。在此基础上,根据已知的模式生物细胞凋亡机理逐步推测线粒体上游途径的参与者(如DNA损伤、p53蛋白、Bcl-2家族蛋白、细胞色素c和caspase-3等),并用相关实验进行验证,最终勾画出较为完整的铅诱导细胞凋亡的线粒体途径。此外,通过检测内质网凋亡事件(如内质网特异性的凋亡酶caspase-12和钙蛋白酶calpain是否被激活;GRP78和CHOP表达是否发生改变等),进一步探索内质网途径在此过程中的作用,以期对铅诱导凋亡的机理有更为全面的认识。主要实验结果:1.乙酸铅能引起PC 12细胞增殖显著下降。2.光镜下观察,乙酸铅对PC 12细胞的形态和细胞骨架无明显改变。3.电镜下观察,乙酸铅能使细胞核固缩,线粒体肿胀、正常结构消失并伴有空泡化,胞内出现鼓泡现象,但内质网未见异常。4.乙酸铅能引起PC 12细胞DNA链断裂,损伤程度存在浓度依赖关系。5.乙酸铅能诱导PC 12细胞发生凋亡,并存在浓度依赖关系。6.乙酸铅能引起caspase-3活化程度显著增强,并存在浓度依赖关系。7.乙酸铅对PC 12细胞的calpain活性无明显改变。8.乙酸铅能促进PC 12细胞胞质细胞色素c水平的提高,Bax和p53表达增加,Bcl-2表达下降,Bax/Bcl-2比值上升。9.乙酸铅能促进PC 12细胞GRP78表达的增加,而CHOP和procaspase-12表达未改变,caspase-12未剪切活化。结论:1.铅诱导细胞凋亡的线粒体途径:铅通过损伤细胞DNA诱导p53激活,并进一步引起Bax表达增加,导致Bax/Bcl-2比值升高,线粒体通透性改变,细胞色素c等促凋亡物质释放入胞质,引起caspase级联激活,并最终导致细胞凋亡的发生。2.结合形态学和生化研究结果,在本研究条件下,铅通过线粒体途径诱导细胞凋亡,而内质网途径可能并未参与此过程。3.铅诱导细胞凋亡生化指标改变的同时,细胞器也发生了形态学改变。4.细胞凋亡可能在铅致毒过程中起了重要的作用。更多还原

【Abstract】 Lead is a common kind of heavy metal, and it is widely used in industry and daily lives because of its malleability, low melting point and resistance to corrosion. As an unbiodegradable material, lead exists persistently in all phases of environment and biological systems, therefore causes global contaminations of air, water and soil, and presents a great threat to human health. Lead exposure can induce a wide range of physiological, biochemical and behavioral dysfunctions in human beings. A variety of researches showed that lead could cause toxic effects to almost all human systems, including nervous system, hematological system, cardiovascular system, urinary system, reproductive system, skeletal system, endocrine system, and immune system. Due to the severe damage to the mental development of preschool children, neurotoxicity of lead gained even more attention.The molecular mechanisms of lead toxicity have been well eclucidated in many previous studies, and it is presumed that lead may exert toxicity through its affinity with certain proteins and its ability to mimic calcium. Recent studies also found that lead could induce apoptosis in rat brain, testis, fibroblasts, lung, and retinal rod cells, which indicated that apoptosis might also play an important role in the lead toxicity. Apoptosis is a highly controlled process in which cell death is executed to maintain the steady state under physiological conditions and respond to various stimuli. The regulations of apoptosis are complex, involving three important pathways: death receptor pathway, mitochondrial pathway, and endoplasmic reticulum pathway. Hundreds of genes and proteins are involed in these pathways and accordingly form a network to regulate apoptosis precisely. In lead-induced apoptosis, it is widely accepted that the mitochondria are most pertinent in mediating apoptosis. However, it is still unclear how lead induces mitochondrial dysfunction and which factors are involved in the upstream of mitochondrial pathway. Furthermore, the fact that lead can disturb intracellular Ca²⁺ homeostasis also leads us to investigate the involvement of endoplasmic reticulum pathway in lead-induced apoptosis.In summary, the present study used PC 12 cell line, a regular model for neuron study, to confirm whether lead could induce apotosis. Moreover, according to the known apoptotic mechanisms gained from model organisms, we speculated the possible participants of upstream mitochondrial pathway （such as DNA damage, p53, Bcl-2 family, cytochrome c and caspase-3, etc.） and tried to identify them through a series of experiments. Therefore we might draw a relatively complete picture of the mitochondrion-mediated apoptotic pathway. Besides, the endoplasmic reticulum events （such as the activations of caspase-12 and calpain, and overexpressions of CHOP and GRP 78） were measured to explore whether endoplasmic reticulum played an important role in this process, which could give us a more comprehensive understanding of apoptotic mechanism induced by lead.The main results: 1. Lead acetate decreased the proliferation of PC 12 cells.2. Under light microscope, lead acetate had no effect on the cell morphology and cytoskeleton.3. Under electron microscope, lead acetate induced nuclear pyknosis, mitochondrial swelling and vacuolization, cell blebbing. But the structure of endoplasmic reticulum remained unchanged.4. Lead acetate induced DNA damage with a concentration-dependent manner in PC 12 cells.5. Lead acetate induced apoptosis with a concentration-dependent manner in PC 12 cells.6. Lead acetate increased the activation level of caspase-3 with a concentration-dependent manner in PC 12 cells.7. Lead acetate had no effect on the activity of calpain in PC 12 cells.8. Lead acetate increased cytosolic cytochrome c level and the expressions of Bax and p53. The expression of Bcl-2 decreased, and the ratio of Bax/Bcl-2 increased significantly.9. Lead acetate increased the expression of GRP78, but the expressions of CHOP and procaspase-12 remained unchanged. Furthermore, caspase-12 wasn’t activated.Conclusions:1. The possible mitochondrial pathway of lead-induced apoptosis: Lead can induce DNA damage, which may activate p53. After the ratio of Bax/Bcl-2 increases, the apoptotic factors （such as cytochrome c） are released from mitochondria, which induce caspases cascade activation and apoptosis.2. The morphiological and biochemical results indicate that under the conditions of this research, lead may induce apoptosis through mitochondrial pathway, but not endoplasmic reticulum pathway.3. Accompanied with the lead-induced apoptotic biochemical alterations, the morphology of organelle also changes.4. Apoptosis may play an important role in lead toxicity.更多还原