铁过载引发人肝细胞损伤的机制研究
发布时间：2018-11-03 12:28
【摘要】：骨髓增生异常综合症(Myeloidysplastic Syndrome,MDS)是一组起源于造血髓系定向干细胞或多能干细胞的异质性克隆性疾患。在我国,MDS发病率有很明显上升趋势,发病率已达到十万分之三,且患者发病年龄比西方国家年轻十岁左右。治疗MDS的主要手段是输血或造血干细胞移植。越来越多的证据发现接受造血干细胞移植后,病人的肝脏出现典型的铁过载症状进而引起病人肝损伤。此外,铁过载还抑制了病患的造血功能及治疗后恢复。尽管当前临床上利用祛铁胺(Desferrioxamine,DFO)、祛铁酮(Deferiprone,DFP)和地拉罗司(Deferasirox,DFX或ICL670)等祛铁药物能够降低肝实质细胞中的铁水平、保护脏器器官并改善MDS患者的预后,但是对于病患祛铁药物治疗所使用的合适剂量仍充满争议,同时祛铁治疗过程漫长且昂贵。因此,深入研究铁过载损伤的分子机制对病患铁过载的治疗具有重要意义。本课题组以人肝细胞HH4为研究模型,详细研究了体外铁过载培养条件下细胞凋亡发生、基因Hepcidin表达调控的信号通路以及蛋白质组和磷酸化蛋白质组差异表达,主要内容如下:(1)铁过载诱导细胞凋亡发生的机制研究。枸橼酸铁铵(FAC)处理HH4细胞0-72hr后,对胞内铁离子含量、细胞活力、活性氧(ROS)水平、氧化应激蛋白表达变化及细胞凋亡进行考察。结果表明HH4细胞铁过载后,胞内铁离子浓度呈时间依赖性增加,而细胞活力呈现浓度和时间依赖性降低。同时,铁过载处理显著增加胞内ROS水平,而添加抗氧化剂谷胱甘肽(GSH)和N-乙酰半胱氨酸(NAC)可以明显降低FAC诱导的胞内ROS生成。此外,铁过载导致蛋白IκB-α、p38 MAPK和NF-κB p65磷酸化水平增加,以及促进转录因子NF-κB p65入核。实验同时发现铁过载处理导致凋亡相关基因表达水平增加,且流式检测确认凋亡比率显著增高。而对凋亡相关蛋白Fas和Bid进行siRNA瞬间干扰后,细胞活力得到恢复。此外,添加GSH后,细胞凋亡率明显降低且氧化应激活化信号途径和凋亡信号途径相关蛋白如p-IκB-α、p-p38 MAPK、p-NF-κB、Caspase-8、Cytc和Caspase-3表达水平呈现显著下调。本部分实验结果表明铁过载诱导了ROS介导的HH4细胞死亡受体途径及线粒体途径凋亡发生。(2)NF-κB参与Hepcidin表达调控研究。本部分实验以不同浓度FAC(0.1、1、5和10 mmol/L)处理HH4细胞48 hr,半定量PCR法检测胞内Hepcidin表达水平。同时利用染色质免疫共沉淀(ChIP)、电泳迁移率实验(EMSA)和双荧光素酶报告基因系统实验,并结合胞内NF-κB活性抑制实验,确定NF-κB对人Hepcidin转录活性的影响。结果表明5 mmol/L和10 mmol/L FAC处理HH4细胞后,Hepcidin表达水平显著增强。ChIP和EMSA实验证实NF-κB能够与Hepcidin基因启动子结合。重组荧光素酶报告质粒TOPFlash-pHepcidin相对荧光素酶活性明显高于对照组;与重组质粒TOPFlash-pHepcidin相比,突变重组荧光素酶报告质粒TOPFlash-pHepcidin-mut相对荧光素酶活性显著降低。同时NF-κB抑制剂BAY 11-7082显著降低了Hepcidin基因的表达。本部分实验结果表明转录因子NF-κB参与了FAC过载诱导Hepcidin表达上调。(3)肝细胞在铁过载模型中的蛋白质组学研究。借助蛋白质组学技术,我们发现与对照组相比,铁过载处理导致HH4细胞58种蛋白质表达水平显著上调以及35种蛋白质表达水平显著下调。随后的GO分析表明铁过载诱导的93种差异表达蛋白与内吞作用、二价、三价金属阳离子稳态平衡、创伤反应、炎症反应、细胞因子分泌的正调控反应、生长调控、抗凋亡以及凋亡的线粒体改变等生物学过程紧密相关。此外,蛋白质组学实验结果显示FAC过载处理诱发TLR2蛋白表达水平上调7倍和IL6ST蛋白表达水平上调2.9倍,而随后的蛋白质免疫印迹实验和TLR2基因干扰实验证实铁过载处理激活了HH4细胞TLR2-MyD88-p-NF-κB-IL-6炎症路径。而另一方面,磷酸化蛋白质组学实验表明HH4细胞铁过载处理导致335种磷酸化蛋白出现差异变化,其中高达11%的磷酸化蛋白与细胞周期进程有关。磷酸化蛋白质组学实验结果同时显示HH4细胞铁过载处理后G2/M期转换关键蛋白CDK1在Thr14和Tyr15位点磷酸化修饰水平增高约10.9倍。进一步的流式检测证实铁过载能够引发HH4和小鼠肝细胞NMH细胞G2/M期阻滞,同时RT-PCR和蛋白免疫印迹检测则表明FAC过载可能经由p53-p21-CDK1、p53-14-3-3 sigma-CDK1或14-3-3 gamma路径诱导了HH4细胞G2/M期阻滞发生。上述实验结果表明铁过载处理导致HH4细胞TLR2介导的炎症反应发生及G2/M期阻滞。
[Abstract]:Myelodysplastic Syndromes (MDS) is a group of heterogeneous clonal disorders derived from hematopoietic stem cells or pluripotent stem cells. In China, the incidence of MDS increased significantly, and the incidence of MDS was up to ten thousand three ter, and the incidence of MDS was about ten years younger than that of Western countries. The main means of treating MDS are transfusion or hemopoietic stem cell transplantation. More and more evidence has found that after transplantation of hematopoietic stem cells, a typical iron overload condition in the liver of a patient leads to a patient's liver injury. In addition, iron overload inhibits the hematopoietic function and post-treatment recovery of the patient. In spite of the current clinical use of desferrioxamine (DFO), deferione (DFP) and deersirox (DFX or ICL670), iron-removing agents can reduce iron levels in liver parenchyma cells, protect organ organs and improve the prognosis of MDS patients, but the appropriate dosage used for the treatment of patients with iron therapy is still controversial while removing iron treatment is a long and expensive process. Therefore, in-depth study of the molecular mechanism of iron overload injury is of great significance to the treatment of iron overload in patients. In this study, human hepatocyte HH4 was used as the research model, the cell apoptosis occurred under the condition of iron overload culture in vitro, the signal pathway of the expression and regulation of gene Hepcidin, and the differential expression of protein group and phosphorylated protein group were studied in detail. The main contents were as follows: (1) The mechanism of iron overload induced apoptosis. The intracellular iron ion content, cell viability, reactive oxygen species (ROS) levels, oxidative stress protein expression and apoptosis were investigated after treatment of HH4 cells for 0-72hr with iron disulfide (FAC). The results showed that the concentration of iron ions increased in the cytoplasm of HH4 cells, while the concentration and time dependence of cell viability decreased. At the same time, iron overload treatment significantly increased intracellular ROS level, while addition of antioxidant glutathione (GSH) and N-cysteine (NAC) could significantly reduce the intracellular ROS generation induced by FAC. In addition, iron overload results in increased phosphorylation of protein I, B-, p38 MAPK, and NF-NADB p65, as well as promoting transcription factor NF-NADB p65 into the nucleus. At the same time, it was found that iron overload treatment resulted in an increase in the expression level of apoptosis-related genes, and the rate of apoptosis was significantly increased. The apoptosis-related proteins Fas and Bid were transiently disrupted by siRNA, and the viability of the cells was restored. In addition, after addition of GSH, the cell apoptosis rate was decreased significantly and oxidative stress activated signaling pathway and apoptosis signal pathway related proteins, such as p-I, B-, p-p38 MAPK, p-NF-Sepharose B, Caspase-8, Cytc and Caspase-3, exhibited a significant downregulation. The results of this part suggest that iron overload induces ROS-mediated HH4 cell death receptor pathway and mitochondrial pathway apoptosis. (2) NF-Sepharose B was involved in the study of the expression and regulation of HepcGVHD. HH4 cells were treated with different concentrations of FAC (0. 1, 1, 5 and 10 mmol/ L) for 48 hr, and the expression level of Hepcidin was detected by semi-quantitative PCR. The effect of NF-Sepharose B on the transcription activity of human Hepci2 was determined by means of chromatin immune co-precipitation (ChIP), electrophoretic mobility assay (EMSA) and double luciferase reporter gene system. The results showed that after treatment of HH4 cells with 5 mmol/ L and 10 mmol/ L FAC, the expression level of HepcGVHD was significantly enhanced. The ChIP and EMSA experiments confirmed that NF-Sepharose B was able to bind to the Hepcidin gene promoter. The relative luciferase activity of the recombinant luciferase reporter plasmid was significantly higher than that of the control group. At the same time, NF-Sepharose B inhibitor BAY 11-7082 significantly reduced the expression of Hepcidin gene. The results of this part show that the transcription factor NF-5B is involved in the upregulation of the expression of FAC overload-induced HepcGVHD. (3) proteomic study of hepatocytes in iron overload models. With the aid of proteomics techniques, we found that iron overload treatment resulted in a significant increase in the expression levels of 58 proteins in HH4 cells and a significant down-regulation of 35 protein expression levels in comparison to the control group. Subsequent GO analysis indicated that 93 differentially expressed proteins induced by iron overload were regulated by positive regulation and growth regulation, such as endocytic action, bivalent, trivalent metal cation homeostasis, wound response, inflammatory response, cytokine secretion, Anti-apoptosis and apoptosis are closely related to biological processes such as mitochondrial change. In addition, the protein group learning experiment showed that the expression level of TLR2 protein increased by 2.7 times and IL6ST protein expression level increased by 2.9 times than that of the FAC overload treatment, while the subsequent Western blot experiment and the TLR2 gene interference experiment confirmed that the iron overload treatment activated the HH4 cell TLR2-MyD88-p-NF-Sepharose B-IL-6 inflammatory pathway. On the other hand, the phosphorylation protein group learning experiment indicated that the HH4 cell iron overload treatment resulted in the variation of 335 phosphorylation proteins, among which 11% of the phosphorylated proteins were related to the cell cycle progression. At the same time, the phosphorylation protein CDK1 in the G2/ M phase after the overload treatment of HH4 cells was increased by about 10. 9 times higher than that in Thr14 and Tyr15 sites. Further flow detection confirmed that iron overload could induce the G2/ M phase arrest of HH4 and mouse liver cells, while RT-PCR and Western blot analysis showed that FAC overload could induce the G2/ M block of HH4 cells via p53-p21-CDK1, p53-14-3-3sigma-CDK1 or 14-3-3 pathway. The results of the above experiment indicated that the iron overload treatment resulted in the inflammation response mediated by TLR2 and G2/ M arrest in HH4 cells.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R551.3
[Abstract]:Myelodysplastic Syndromes (MDS) is a group of heterogeneous clonal disorders derived from hematopoietic stem cells or pluripotent stem cells. In China, the incidence of MDS increased significantly, and the incidence of MDS was up to ten thousand three ter, and the incidence of MDS was about ten years younger than that of Western countries. The main means of treating MDS are transfusion or hemopoietic stem cell transplantation. More and more evidence has found that after transplantation of hematopoietic stem cells, a typical iron overload condition in the liver of a patient leads to a patient's liver injury. In addition, iron overload inhibits the hematopoietic function and post-treatment recovery of the patient. In spite of the current clinical use of desferrioxamine (DFO), deferione (DFP) and deersirox (DFX or ICL670), iron-removing agents can reduce iron levels in liver parenchyma cells, protect organ organs and improve the prognosis of MDS patients, but the appropriate dosage used for the treatment of patients with iron therapy is still controversial while removing iron treatment is a long and expensive process. Therefore, in-depth study of the molecular mechanism of iron overload injury is of great significance to the treatment of iron overload in patients. In this study, human hepatocyte HH4 was used as the research model, the cell apoptosis occurred under the condition of iron overload culture in vitro, the signal pathway of the expression and regulation of gene Hepcidin, and the differential expression of protein group and phosphorylated protein group were studied in detail. The main contents were as follows: (1) The mechanism of iron overload induced apoptosis. The intracellular iron ion content, cell viability, reactive oxygen species (ROS) levels, oxidative stress protein expression and apoptosis were investigated after treatment of HH4 cells for 0-72hr with iron disulfide (FAC). The results showed that the concentration of iron ions increased in the cytoplasm of HH4 cells, while the concentration and time dependence of cell viability decreased. At the same time, iron overload treatment significantly increased intracellular ROS level, while addition of antioxidant glutathione (GSH) and N-cysteine (NAC) could significantly reduce the intracellular ROS generation induced by FAC. In addition, iron overload results in increased phosphorylation of protein I, B-, p38 MAPK, and NF-NADB p65, as well as promoting transcription factor NF-NADB p65 into the nucleus. At the same time, it was found that iron overload treatment resulted in an increase in the expression level of apoptosis-related genes, and the rate of apoptosis was significantly increased. The apoptosis-related proteins Fas and Bid were transiently disrupted by siRNA, and the viability of the cells was restored. In addition, after addition of GSH, the cell apoptosis rate was decreased significantly and oxidative stress activated signaling pathway and apoptosis signal pathway related proteins, such as p-I, B-, p-p38 MAPK, p-NF-Sepharose B, Caspase-8, Cytc and Caspase-3, exhibited a significant downregulation. The results of this part suggest that iron overload induces ROS-mediated HH4 cell death receptor pathway and mitochondrial pathway apoptosis. (2) NF-Sepharose B was involved in the study of the expression and regulation of HepcGVHD. HH4 cells were treated with different concentrations of FAC (0. 1, 1, 5 and 10 mmol/ L) for 48 hr, and the expression level of Hepcidin was detected by semi-quantitative PCR. The effect of NF-Sepharose B on the transcription activity of human Hepci2 was determined by means of chromatin immune co-precipitation (ChIP), electrophoretic mobility assay (EMSA) and double luciferase reporter gene system. The results showed that after treatment of HH4 cells with 5 mmol/ L and 10 mmol/ L FAC, the expression level of HepcGVHD was significantly enhanced. The ChIP and EMSA experiments confirmed that NF-Sepharose B was able to bind to the Hepcidin gene promoter. The relative luciferase activity of the recombinant luciferase reporter plasmid was significantly higher than that of the control group. At the same time, NF-Sepharose B inhibitor BAY 11-7082 significantly reduced the expression of Hepcidin gene. The results of this part show that the transcription factor NF-5B is involved in the upregulation of the expression of FAC overload-induced HepcGVHD. (3) proteomic study of hepatocytes in iron overload models. With the aid of proteomics techniques, we found that iron overload treatment resulted in a significant increase in the expression levels of 58 proteins in HH4 cells and a significant down-regulation of 35 protein expression levels in comparison to the control group. Subsequent GO analysis indicated that 93 differentially expressed proteins induced by iron overload were regulated by positive regulation and growth regulation, such as endocytic action, bivalent, trivalent metal cation homeostasis, wound response, inflammatory response, cytokine secretion, Anti-apoptosis and apoptosis are closely related to biological processes such as mitochondrial change. In addition, the protein group learning experiment showed that the expression level of TLR2 protein increased by 2.7 times and IL6ST protein expression level increased by 2.9 times than that of the FAC overload treatment, while the subsequent Western blot experiment and the TLR2 gene interference experiment confirmed that the iron overload treatment activated the HH4 cell TLR2-MyD88-p-NF-Sepharose B-IL-6 inflammatory pathway. On the other hand, the phosphorylation protein group learning experiment indicated that the HH4 cell iron overload treatment resulted in the variation of 335 phosphorylation proteins, among which 11% of the phosphorylated proteins were related to the cell cycle progression. At the same time, the phosphorylation protein CDK1 in the G2/ M phase after the overload treatment of HH4 cells was increased by about 10. 9 times higher than that in Thr14 and Tyr15 sites. Further flow detection confirmed that iron overload could induce the G2/ M phase arrest of HH4 and mouse liver cells, while RT-PCR and Western blot analysis showed that FAC overload could induce the G2/ M block of HH4 cells via p53-p21-CDK1, p53-14-3-3sigma-CDK1 or 14-3-3 pathway. The results of the above experiment indicated that the iron overload treatment resulted in the inflammation response mediated by TLR2 and G2/ M arrest in HH4 cells.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R551.3
【相似文献】
相关期刊论文 前10条
1 李瀚e，
本文编号：2307787
本文链接：https://www.wllwen.com/yixuelunwen/xxg/2307787.html
最近更新
教材专著
