【作者】 汪纪仓；

【导师】 刘宗平；

【作者基本信息】 扬州大学 ， 临床兽医学， 2010， 博士

【摘要】 镉是许多工业广泛使用的高毒重金属环境污染物,镉对人和其他哺乳动物的肝、肾、肺、胰腺、睾丸、胎盘和骨等许多器官和组织都有毒性。镉所致机体损伤的机制十分复杂且尚不明确,体内外实验研究证明镉可引起细胞氧化应激或凋亡/死亡,且凋亡与线粒体、细胞内钙离子动态平衡、丝裂原活化蛋白激酶(MAPK)的激活等有关。对啮齿动物研究证明,镉最初主要累积在肝脏,所以暴露毒性剂量的镉首先引起肝脏受损。本论文以大鼠肝细胞为模型,采用细胞生物学和分子生物学的方法研究镉致体外培养大鼠肝细胞的毒性作用及机理。1.镉对大鼠肝细胞毒性损伤的研究采用两步灌流法获得大鼠肝细胞,经过24h培养,换以含2.5、5、10μmol/L醋酸镉的无血清培养基培养12h和24h,测定镉对细胞存活率、细胞培养上清液中LDH、ALT和AST的活性,以及镉对细胞形态、超微结构的影响。结果表明,随着镉浓度的增大,肝细胞的存活率逐渐降低,12h和24h时各剂量染镉组细胞存活率均极显著低于对照组(P<0.01)；随着镉剂量的增加和作用时间的延长,细胞急剧变形,坏死细胞增多,线粒体肿胀变形,线粒体嵴模糊,内容物皱缩空泡化或最终崩解。LDH、AST和ALT释放量随剂量增加而增加,部分剂量组LDH、AST和ALT活性与对照组相比差异显著或极显著(P<0.05或P<0.01)。2.镉对大鼠肝细胞凋亡及氧化损伤的研究醋酸镉处理肝细胞,测定镉对细胞凋亡率、氧化应激指标、活性氧(ROS)水平及线粒体膜电位(△Ψm)的变化。结果表明,12h时细胞内GSH含量随镉剂量增高而降低,部分剂量组极显著(P<0.01)低于对照组,24h时随剂量增高而升高,5和10μmol/L剂量组显著或极显著(P<0.01)高于对照组,GSH-PX的活性变化与之相反；随着镉剂量的增大和作用时间延长,SOD和CAT活性、MDA含量均升高,部分剂量与对照组相比差异显著或极显著(P<0.05或P<0.01)；而GST和GR活性降低,但与对照组无明显差异(P>0.05)；细胞凋亡率升高,细胞内ROS水平增加和△Ψm降低,部分剂量组与对照组差异显著或极显著(P<0.05或P<0.01)；表明镉可致肝细胞产生氧化应激,且通过线粒体途径引起细胞凋亡。3.N-乙酰半胱氨酸对镉致肝细胞损伤的影响对肝细胞染毒的同时添加NAC,通过测定肝细胞的存活率、细胞形态和凋亡、△甲m和ROS的变化,观察NAC对镉致大鼠肝细胞损伤的保护作用。结果表明,NAC可显著或极显著提高镉处理细胞存活率(P<0.05或P<0.01),呈剂量-效应关系,NAC可使镉处理组皱缩和坏死细胞、凋亡细胞明显减少,显著或极显著降低镉处理细胞ROS生成和阻止△Ψm的降低(P<0.05或P<0.01)。表明NAC可有效保护镉致大鼠肝细胞的毒性损伤。4.镉致大鼠肝细胞凋亡的非caspase途径用醋酸镉处理肝细胞,观察caspase-3含量变化以及caspases广谱抑制剂Z-VAD-fink对镉致细胞存活率、形态和凋亡变化的影响。结果表明,镉暴露的不同时间,肝细胞caspase-3活性无明显变化,与对照组差异不显著(P>0.05); Z-VAD-fmk对镉所致的细胞存活率、细胞形态和细胞凋亡无显著影响。表明caspase途径未参与镉致大鼠肝细胞的凋亡过程。5.钙离子超载在镉致大鼠肝细胞凋亡中的作用通过不同浓度醋酸镉处理肝细胞,不同时间点测定细胞内钙离子浓度,以及钙离子抑制剂Bapta-AM对镉致细胞内钙离子浓度、细胞存活率、细胞形态、细胞凋亡、细胞内ROS和△Ψm变化的影响。结果表明,镉暴露1.5h时细胞内钙离子水平极显著升高(P<0.01),但随着时间的延长,细胞内钙离子水平迅速下降,并接近对照组水平。而Bapta-AM可以显著降低镉引起的钙离子水平升高,并显著提高镉处理细胞的存活率(P<0.05)。另外Bapta-AM可使镉处理细胞形态趋于完整、并使变形细胞和凋亡细胞减少,显著或极显著减少ROS产生和抑制△Ψm降低(P<0.05或P<0.01)。说明镉可致肝细胞内钙离子水平升高,钙离子抑制剂Bapta-AM能有效降低细胞内钙离子水平及细胞损伤,提示钙离子超载在镉致肝细胞凋亡中发挥重要作用。6.镉致大鼠肝细胞凋亡的MAPK途径在10μmol/L醋酸镉处理肝细胞的同时,用p38抑制剂SB202190、JNK抑制剂SP600125、ERK抑制剂U0126单独或联合作用于肝细胞,测定细胞存活率、细胞形态、细胞凋亡率、△甲m和磷酸化p38蛋白表达的变化。结果表明,SB202190可以显著或极显著提高镉处理组细胞的存活率,减少变形细胞和凋亡细胞数量(P<0.05或P<0.01),但SP600125和U0126作用相反。镉可极显著提高肝细胞磷酸化p38的表达量(P<0.01),而SB202190能极显著降低其表达(P<0.01)；SB202190还可极显著升高镉引起的△甲m降低(P<0.01)。说明镉暴露使肝细胞产生的ROS引起p38 MAPK途径激活,并通过损伤线粒体引起细胞凋亡。更多还原

【Abstract】 Cadmium (Cd), an extremely important environment pollutant, was widely used in many industries. In human and other mammals, Cd affects adversely a number of organs and tissues, including the liver, kidney, lung, pancreas, testis, placenta and bone. Cd induced injury is very complex and unclear in the body. Cd induced cellular oxidative stress or apoptosis/death in vitro and in vivo. Apoptosis has relationship with mitochondria, intracellular calcium homeostasis, mitogen-activated protein kinase (MAPK) activitied and so on. Cd mainly accumulate in the liver initialy through rodent studies, so exposure to toxic doses of Cd will cause liver damage first. Therefore, we choose rat hepatocytes to study the mechanism of Cd induced cytotoxicity with cell biology and molecular biology method.1. Cd-induced cytotoxicity in rat hepatocytesRat hepatocytes were isolated by a two-step perfusion technique. After 24h planting, hepatocytes were treated with cadmium acetate (Cd(AC)2) (2.5,5 and 10μmol/L) for 12 h or 24 h. Cell viability, the leakage of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT) in cell-free medium were measured with MTT assay and spectro photometric assay at the end of incubation. Hepatocytes morphology and ultrastructure were evaluated.The results showed that with the dose of Cd increased and long expored, hepatocytes viability was decreased very significantly (P＜0.01) after 12 h or 24 h treatment, hepatocytes showed sharp deformation and necrosis, mitochondria swelling and degeneration, mitochondrial cristae blurred, deformed or final collapse.The leakage of LDH, ALT and AST increased with doses increased, there was significant difference in partial groups than the control group (P＜0.05 or P<0.01). 2. Oxidative stress induced by Cd triggers apoptosis in rat hepatocytes24 h rat hepatocytes were treated with Cd(AC)2. The effects of Cd on oxidative stress markers, apoptosis, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm) collapse were measured.The results showed that the GSH content decreased with the dose of Cd increased, there was very significant difference in partial groups than the control group (P<0.01) at 12 h, but GSH content increased with the dose of Cd increased at 24 h. GSH-PX activities was in opposition with GSH content. The activities of CAT and SOD and MDA level increased, there was significant difference in partial groups than the control group (P＜0.05 or P＜0.01). The GST and GR activities were decreased but have no significant change. Cd induced apoptosis rate increased,ΔΨm collapse and ROS generation. There was significant difference in partial groups than the control group (P<0.05 or P<0.01).These results showed that Cd induces ROS generation and oxidative stress. Cd induces apoptosis through mitochondrial pathway.3. The effect of NAC on Cd induced cytotoxicityRat hepatocytes were treated with Cd in the presence or absence of NAC. The effects of NAC on Cd-induced cell morphology, apoptosis, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm) collapse were measured.These results showed that NAC significantly prevented hepatocytes from Cd-induced death (P＜0.05 or P＜0.01). NAC effectively inhibited Cd-induced alterations in the morphology of hepatocytes. NAC insult effectively protected hepatocytes against apoptosis from Cd. Cd induce ROS generation andΔΨm collapse were blocked by NAC. It showed that NAC can protect Cd-induced cytotoxicity.4. Cd-induced apoptosis in rat hepatocytes through caspase-independent way24 h rat hepatocytes were treated with Cd. Caspase-3 activities was measured. The effects of Z-VAD-fmk on Cd-induced cell viability, morphology and apoptosis in hepatocytes were evaluated. The results showed that caspase-3 activity does not increase, Z-VAD-fmk has no effect on cell survival, morphology and apoptosis. It showed that Cd induced hepatocytes apoptosis involvement of caspase-independent pathway.S.Involvement of calcium in Cd-induced apoptosis in rat hepatocytesHepatocytes were treated with Cd for certain time, then intracellular calcium concentration were determinated. And calcium chelator Bapta-AM on the impact of intracellular calcium concentration, cell survival, morphology and apoptosis, ROS generation andΔΨm collapse were evaluated.The results showed the level of intracellular calcium was very significantly increased induced by Cd at 1.5 h (P<0.01). However, with time increasing, the level of intracellular calcium levels rapidly decreased to the level of control group. Bapta-AM can attenuate the level of calcium and significant increase the cell viability induced by Cd (P＜0.05). Bapta-AM effectively inhibited Cd-induced alterations in the morphology of hepatocytes. Bapta-AM insult effectively protected hepatocytes against apoptosis from Cd. Cd induce ROS generation andΔΨm collapse were blocked by Bapta-AM. It showed that Cd can elevate intracellular calcium levels, then induce apoptosis through mitochondrial pathway.6.Cd induced apoptosis throuth p38 MAPK in rat hepatocytesHepatocytes were treated with Cd in the presence or absence of MAPK signaling inhibitors (p38 MAPK inhibitor SB202190, JNK inhibitor SP600125, ERK inhibitor U0126), then their effect on cell viability, morphology and apoptosis were investigated. Immunohistochemistry analysis was performed to recognize the activated phosphorylated forms of p38 MAPK kinases in Cd-treated hepatocytes in the presence or absence SB202190 and NAC. The impact of SB202190 on Cd-inducedΔΨm collapse was resurched.The results showed that SB202190 reversed significantly Cd-induced cell death. SB202190 effectively inhibited Cd-induced alterations in the morphology of hepatocytes. SB202190 protected hepatocytes against apoptosis from Cd. While SP600125 and U0126 increased Cd-induced cell death significantly. The phosphorylation of p38 MAPK increased after Cd treatment and these activations were inhibated by the treatment with its inhibitor SB202190 or NAC. SB202190 blocked disruption ofΔΨm. The findings suggested that Cd induction of ROS activated the p38 MAPK pathway, triggering apoptosis through mitochondrial pathway.更多还原