【作者】 张义冉；

【导师】 刘宗平；

【作者基本信息】 扬州大学 ， 临床兽医学， 2013， 博士

【摘要】 镉(cadmium, Cd)是一种有毒的重金属元素,易在体内蓄积,镉能够诱导多种器官或组织损伤并导致相关功能丧失,包括肾脏、肝脏、肺脏、骨骼、心血管系统和免疫系统。体内外研究均证明,镉可引起细胞凋亡、氧化损伤及DNA损伤,且凋亡与氧化应激、线粒体损伤、促分裂原激活蛋白激酶(MAPK)、死亡受体、Caspase及非Caspase依赖性通路的激活密切相关。肝脏是镉损伤的主要靶器官之一,目前对镉致肝细胞损伤研究较多,但其作用机制还不完全清楚。本研究以大鼠肝细胞系BRL3A细胞为模型,采用细胞生物学和分子生物学方法研究镉对BRL3A细胞的毒性作用机制及NAC的保护作用。研究内容如下：1.镉致BRL3A细胞毒性损伤及NAC的保护作用为研究镉致BRL3A细胞的毒性损伤及NAC的保护作用,以0、10、20、40μmol/L醋酸镉分别作用BRL3A细胞12h(分别为a-d组),同时以2mmol/LNAC、2mmol/L NAC(预孵育30min)+20μmol/L醋酸镉分别作用BRL3A细胞12h(为e-f组)。倒置显微镜观察细胞形态的变化,采用MTT法测定细胞存活率,比色法测定培养上清中LDH活性。结果表明,随着镉浓度的增加,细胞皱缩、变圆、结构不完整,细胞存活率逐渐降低(p<0.01),LDH释放量显著增加(p<0.05); NAC单独处理组细胞形态、细胞存活率和LDH释放量与对照组差异不显著(p>0.05); NAC与镉联合处理组细胞形态皱缩,细胞存活率极显著升高(p<0.01),上清中LDH活性极显著降低(p<0.01)。表明NAC可有效保护镉致BRL3A细胞的毒性损伤。2.镉致BRL3A细胞凋亡及NAC的保护作用为研究镉对BRL3A细胞凋亡的影响及NAC的保护作用,以0、10、20、40μmol/L醋酸镉分别作用BRL3A细胞12h(分别为a-d组),同时以2mmol/L NAC、2mmol/L NAC(预孵育30min)+20μmol/L醋酸镉分别作用BRL3A细胞12h(为e-f组)。Hoechst33258荧光染色观察细胞核的形态变化,流式细胞仪测定细胞凋亡率。结果表明,随着镉浓度的增加,细胞染色质浓缩,有的呈新月形,甚至出现核碎裂；镉致BRL3A细胞凋亡率显著或极显著升高(p<0.01或p<0.05)；NAC单独处理组细胞核状态和凋亡率均与对照组差异不显著(p>0.05),NAC与镉联合处理组细胞凋亡形态有所减少,凋亡率极显著降低(p<0.01)。表明NAC可有效降低镉致BRL3A细胞的凋亡。3.镉对BRL3A细胞I)NA损伤的研究为研究镉致BRL3A细胞的DNA损伤,以0、10、20、40μmol/L醋酸镉分别作用BRL3A细胞12h(分别为a-d组),通过单细胞凝胶电泳法测定细胞DNA损伤。结果表明,随着镉浓度的增加,受损细胞彗星率、拖尾长度、尾部DNA含量均显著或极显著升高(p<0.01或p<0.05)。表明镉可引起BRL3A细胞DNA损伤,并呈剂量—效应关系。4.镉致BRL3A细胞氧化损伤及NAC的保护作用为研究镉致BRL3A细胞的氧化损伤及NAC的保护作用,以0、10.20、40μmol/L醋酸镉分别作用BRL3A细胞12h(分别为a-d组),同时以2mmol/L NAC、2mmol/L NAC(预孵育30min)+20μmol/L醋酸镉分别作用BRL3A细胞12h(为e-f组)。流式细胞术检测细胞内ROS水平,比色法检测MDA含量及SOD、GSH-Px活性的变化。结果表明,随着镉浓度的增加,细胞ROS、MDA含量显著或极显著升高(p<0.01或p<0.05),SOD、GSH-Px活性显著或极显著降低(p<0.01或p<0.05);NAC单独处理组细胞ROS、MDA含量及SOD、 GSH-Px活性均与对照组差异不显著(p>0.05)；NAC与镉联合处理组细胞ROS含量显著降低(p<0.05), SOD、GSH-Px活性显著或极显著增高(p<0.01或p<0.05)。表明镉致BRL3A细胞发生氧化损伤,NAC具有明显的效保作用。5.镉致BRL3A细胞线粒体损伤及NAC的保护作用为研究镉致BRL3A细胞线粒体损伤及NAC的保护作用,以0、10、20、40μmol/L醋酸镉分别作用BRL3A细胞1213(分别为a-d组),同时以2mmol/LNAC、2mmol/L NAC(预孵育30min)+20μmol/L醋酸镉分别作用BRL3A细胞12h(为e-f组)。透射电子显微镜观察细胞线粒体超微结构的变化,免疫荧光法检测AIF、EndoG蛋白表达,流式细胞术检测线粒体膜电位,比色法检测Caspase3、Caspse9活性的变化,同时用实时荧光定量PCR和免疫印迹法检测了Bax、Bcl-2的mRNA及蛋白,Caspse3、Caspase9及PARP的蛋白表达水平。结果表明,随着镉浓度的增加,线粒体数量减少、肿胀,嵴断裂,发生空泡化,线粒体膜电位均极显著降低(p<0.01),Caspase3、Caspase9活性显著或极显著升高(p<0.01或p<0.05), Bax蛋白及mRNA表达水平均显著增加,Bcl-2蛋白及mRNA表达水平均显著降低(p<0.01Caspase3、Caspase9、PARP的蛋白表达量均显著降低, Cleaved-Caspase3、Cleaved-Caspase9的蛋白表达量则显著增加；NAC单独处理组细胞线粒体结构正常,Bax、Bcl-2、Caspase3、Caspase9、PARP蛋白与对照组无显著差异；NAC的联合应用可有效抑制镉致相关蛋白的变化的趋势。镉可使AIF、EndoG蛋白于线粒体和细胞浆转移入细胞核。表明Caspase和非Caspase途径均参与了镉致BRL3A细胞的凋亡,NAC可有效保护镉致BRL3A细胞的线粒体损伤。6.镉对BRL3A细胞MAPK信号通路的影响及NAC的保护作用为研究镉对BRL3A细胞MAPK信号通路的影响及NAC的保护作用,以0、10、20、40μmol/L醋酸镉分别作用BRL3A细胞12h设为a-d组(分别为a-d组),同时以2mmol/L NAC、2mmol/LNAC(预孵育30min)+20μmol/L醋酸镉分别作用BRL3A细胞12h(为e-f组)。另外,分别用10μmol/Lp38抑制剂SB203580、JNK抑制剂SP600125、ERK抑制剂U0126预孵育0.5h后,加入醋酸镉,使其终浓度20μmol/L作用12h。应用流式细胞术检测细胞凋亡率,免疫印迹法检测MAPK蛋白表达。结果表明,10μmol/L SB203580、 SP600125、U0126处理组细胞凋亡率均极显著降低(p<0.01)。镉处理均能使P-JNK、P-p38的蛋白表达量显著增加,P-ERK在10-20μmol/L染镉组蛋白表达量显著降低,40μmol/L染镉组44kD P-ERK的蛋白磷酸化水平并未显著改变,42kD P-ERK的磷酸化水平则显著增高。表明MAPK途径参与了镉致BRL3A细胞的凋亡,NAC可有效抑制MAPK途径发挥一定的保护作用。7.镉对BRL3A细胞Fas/FasL信号通路的影响及NAC的保护作用为研究镉致BRL3A细胞Fas/FasL信号通路的影响及NAC的保护作用,以0、10、20、40μmol/L醋酸镉分别作用BRL3A细胞12h(分别为a-d组),同时以2mmol/L NAC、2mmol/LNAC(预孵育30min)+20μmol/L醋酸镉分别作用BRL3A细胞12h(为e-f组)。比色法测定Caspase8的活性,免疫印迹法检测Caspase8、FasL蛋白,实时荧光定量PCR检测Fas、FasL mRNA的表达水平。结果表明,随着镉浓度的增加,细胞Caspase8的活性及Cleaved-Caspase8、FasL的蛋白和Fas mRNA的表达水平极显著增加(p<0.01)。FasL mRNA的表达水平则先降低后升高。联合应用NAC可显著抑制镉对Cleaved-Caspase8、FasL蛋白的上调。表明Fas/FasL途径与镉致BRL3A细胞凋亡有关,NAC可有效抑制Fas/FasL途径而发挥一定的保护作用。更多还原

【Abstract】 Cadmium (Cd) is a toxic heavy metal, easy to accumulate in the body. Cd causes a mumber of organs and tissues injury and induces dysfunction, including kidney, liver, lung, bone, cardiovascular system, immune system. Some experiments in vitro and in vivo have already proved that Cd induced cellular apoptosis, oxidatived damage and DNA damage, apoptosis was correlated with oxidative stress, mitochondrial damage, the activation of MAPK, death receptor, Caspase and Caspase independent pathway. Studies revealed that liver is the main target organ of Cd injury. Recently, lots of researches had worked on it, but the mechanism is still not clear. Therefore, we choose the BRL3A immortalized rat hepatocytes to study the mechanism of Cd induced cytotocity and the protection of NAC with cytobiological and molecular biological methods. A series of tests were carried out:1. The cytotoxicity induced by Cd and the protection of NAC in BRL3A cellTo explore the cytotoxicity induced by Cd and the protection of NAC, BRL3A cells were treated with0,10,20, and40μmol/L CdAc2for12h (a-d groups). In the other two experiments, the cells were pre-incubated with2mmol/L NAC for30min and then incubated with20μmol/L CdAc2for12h (e-f groups). The morphology damage was observed by a reverse microscope. Cell viability was evaluated by MTT assay and the leakage of lactate dehydrogenase (LDH) was measured with colorimetric method. The results showed with the increasing dosage of Cd, significant morphological changes showing cell shrinkage, rounding, and loss of cell integrity. Cell viability was decreased significantly (p<0.01), the leakage of LDH increased significantly (p<0.01). NAC alone did not affect the cell morphology, cell viability and the leakage of LDH. Cotreatment with NAC and Cd attenuated morphological changes, increased cell viability and decreased the leakage of LDH significantly (p<0.01). NAC can effectively attenuate the damage induced by Cd in BRL3A cells.2. The effect of apoptotic ratio induced by Cd and the protection of NAC in BRL3A cellTo investigate the effect of apoptosis induced by Cd and the protection of NAC, BRL3A cells were treated with0,10,20, and40μmol/L CdAc2for12h (a-d groups). In the other two experiments, the cells were pre-incubated with2mmol/L NAC for30min and then incubated with20μmol/L CdAc2for12h (e-f groups). BRL3A cells were treated with Cd and NAC for12h, nuclear morphology was analyzed by Hoechst33258staining, cell apoptosis was measured by flow cytometry. The results showed with the increasing dosage of Cd, cell chromatin condensation, cell shrinkage and nuclear fragmentation were observed. The apoptotic ratio increased significantly (p<0.01or p<0.05). NAC alone did not affect the cell nuclear morphology and cell apoptosis. Cotreatment with NAC and Cd attenuated nuclear morphological changes, decreased cell apoptotic ratio significantly (p<0.01). NAC can effectively prohibit the apoptosis induced by Cd in BRL3A cells.3. The DNA damage induced by Cd in BRL3A cellTo observe the DNA damage induced by Cd, BRL3A cells were treated with0,10,20, and40μmol/L CdAc2for12h (a-d groups). DNA damage was measured with single cell gel electrophoresis assay. The results showed with the increasing dosage of Cd, cell commet ratio, tail length and tail DNA percentage increased significantly (p<0.01orp<0.05). Cd induced DNA damage in BRL3A cells.4. The oxidative damage induced by Cd and the protection of NAC in BRL3A cellTo measure the oxidative damage induced by Cd and the protection of NAC, BRL3A cells were treated with0,10,20, and40umol/L CdAc2for12h (a-d groups). In the other two experiments, the cells were pre-incubated with2mmol/L NAC for30min and then incubated with20μmol/L CdAc2for12h (e-f groups). Reactive oxygen species(ROS) was detected by flow cytometry, MDA content and SOD、GSH-Px activity were measured with colorimetric method. The results showed with the increasing dosage of Cd, ROS and MDA content increased significantly (p<0.01orp<0.05), SOD and GSH-Px activity decreased significantly (p<0.01or p<0.05). NAC alone did not affect ROS、MDA content and SOD、GSH-Px activity. Cotreatment with NAC and Cd ROS content decreased significantly (p<0.05), SOD and GSH-Px activity increased significantly (p<0.01orp<0.05). NAC can relieve oxidative damage induced by Cd in BRL3A cell.5. The mitochondrial damage by Cd and the protection of NAC in BRL3A cellTo observe the mitochondrial damage induced by Cd and the protection of NAC, BRL3A cells were treated with0,10,20, and40μmol/L CdAc2for12h (a-d groups). In the other two experiments, the cells were pre-incubated with2mmol/L NAC for30min and then incubated with20μmol/L CdAc2for12h (e-f groups). Mitochondrial ultramicrostructure changes were detected by transmission electron-micriscope and the immunofluorescence of AIF, EndoG were observed, the mitochondrial membrane potential (△Ψm) was detected by flow cytimetry, the activity of Caspase3、Caspse9was detected by colorimetric method, the mRNA and protein level of Bax、Bcl-2, the protein level of Caspse3、Caspase9、PARP were detected by real time fluorescent quantitative PCR and immunoblot. The results showed with the increasing dosage of Cd, mitochondrial swelling and degeneration, mitochondrial cristae blurred, deformed or final collapse, the△Ψm decreased significantly (p<0.01), the activity of Caspase3、Caspse9increased significantly (p<0.01or p<0.05), the mRNA and protein level of Bax increasedand Bcl-2decreased significantly (p<0.01), the protein level of Caspase3、Caspase9、PARP decreased and Cleaved-Caspase3、Cleaved-Caspase9increased. NAC alone did not affect the mitochondrial ultramicrostructure and protein level of Bax、Bcl-2、Caspase3、Caspase9、PARP. Cotreatment with NAC and Cd can inhibit the tendency of the protein level of Bax、Bcl-2、Caspase3、Caspase9、PARP. Cd induced translocation of AIF and EndoG from mitochondria to nucelus. Caspase and Caspase independent pathway toke part in apoptosis induced by Cd in BRL3A cells, NAC can protect the mitochondrial damage induced by Cd in BRL3A cell.6. The effect of Cd on MAPK pathway and the protection of NAC in BRL3A cellTo investigate the effect of MAPK pathway induced by Cd and the protection of NAC, in the first group, BRL3A cells were treated with0,10,20, and40μmol/L CdAc2for12h (a-d groups). In the other two experiments, the cells were pre-incubated with2mmol/L NAC for30min and then incubated with20μmol/L CdAc2for12h (e-f groups). In the second group, BRL3A cells were treated with0,20μmol/L Cd and were pre-incubated with10μmol/L that p38inhibitor (SB203580), JNK inhibitor (SP600125)、ERK inhibitor(U0126) for30min, followed by incubation with20μmol/L Cd for12h, cell apoptotic ratio was detected by flow cytometry and the protein level of MAPK were measured by immunoblot. The results showed the inhibitor of SB203580, SP600125, and U0126decreased cell apoptotic ratio significantly (p<0.01), with the increasing dosage of Cd, the protein level of P-JNK、P-p38increased,10-20μmol/L Cd decreased P-ERK protein level,40μmol/L Cd increased42kD P-ERK protein level,44kD P-ERK protein level didn’t change. MAPK pathway took part in Cd induced BRL3A cell apoptosis and NAC inhibited MAPK pathway.7. The effect of Cd on Fas/FasL pathway and the protection of NAC in BRL3A cellTo investigate the effect of Fas/FasL pathway induced by Cd and the protection of NAC, BRL3A cells were treated with0,10,20, and40μmol/L CdAc2for12h (a-d groups). In the other two experiments, the cells were pre-incubated with2mmol/L NAC for30min and then incubated with20μmol/L CdAc2for12h (e-f groups). The activity of Caspase8was detected by by colorimetric method, the protein level of Caspase8and FasL, the mRNA of Fas、FasL were measured by real time fluorescent quantitative PCR and immunoblot. The results showed with the increasing dosage of Cd, Caspase3activity, the level of Cleaved-Caspase8、FasL protein and Fas mRNA increased significantly (p<0.01), the mRNA level of FasL increased first and then decreased. Fas/FasL pathway is releated with BRL3A cell apoptosis and NAC inhibited Fas/FasL pathway.更多还原