【作者】 李永进；

【导师】 李志超；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2006， 博士

【摘要】 甲基汞环境污染已成为全球性问题,日益引起人们的密切关注。长期接触低剂量甲基汞所致脑发育损伤机制一直是各国学者研究的热点。随着神经分子生物学和生物新技术的迅猛发展,细胞信号通路在神经发育中的作用倍受关注。本研究旨在探讨PKC-MAPK信号通路及PARP在甲基汞所致脑发育毒性中的作用。首先建立接触低剂量甲基汞PC12细胞模型和模拟人体长期接触低剂量甲基汞所致子代脑发育损伤的小鼠仔鼠动物实验模型,分别采用MTT法和流式细胞术(FCM)检测PC12细胞的存活率和凋亡率;应用免疫组织化学、Western blot方法检测PC12细胞和实验动物海马PARP、PKCα表达及ERK、JNK磷酸化水平;利用免疫沉淀法测定PC12细胞和实验动物海马中上述各酶的活性;采用跳台避暗实验检测小鼠仔鼠模型学习记忆能力。结果表明,0.010-0.015μM的甲基汞促进PC12细胞PKCα表达和ERK磷酸化(p<0.05),大于0.020μM的甲基汞则使PC12细胞PKCα表达和ERK磷酸化降低,而JNK磷酸化和PARP表达增强(p<0.05);长期接触含甲基汞1mg/kg的饮水可使仔鼠海马神经元PKCα表达和ERK磷酸化增强,2、4mg/kg实验组则使仔鼠海马PARP表达增强,4mg/kg实验组可使仔鼠海马JNK磷酸化增强,跳台避暗实验结果显示,各实验组小鼠仔鼠的学习记忆能力明显降低(p<0.05)。结论:低剂量甲基汞可影响PC12细胞的分化和存活,诱导PC12细胞凋亡,具有时间和剂量效应关系,并伴有伴有PARP、PKCα表达和ERK、JNK磷酸化双向性改变;接触低剂量甲基汞所致PC12细胞ERK磷酸化的改变可能是通过PKC-MAPK途径实现的;长期接触低剂量甲基汞影响小鼠仔鼠海马神经元PKCα、PARP表达和JNK、ERK磷酸化,降低小鼠仔鼠的学习记忆能力,伴有海马组织超微结构异常改变。本研究可为甲基汞神经发育毒性机制的研究提供重要依据。更多还原

【Abstract】 The worldwide environmental contamination of methylmercury(MeHg) has being a focus from 50th of last centrury. The developing brainappears particularly sensitive to MeHg. Exposure levels in pregnantexperimental animals that do not result in detectable signs or symptomsin the mother can adversely affect the offspring’s development. Studies ofhuman poisonings suggest that may also occur in humans. Humanexposure to MeHg is primarily dietary through the consumption of fish:MeHg is present in all fresh and saltwater fish. Populations that dependon fish as a major source of dietary protein may achieve MeHg exposurelevels hypothesized to adversely affect brain development. Increasingmercury levels in the environment have heightened concerns aboutdietary exposure and a possible role for MeHg in developmentaldisabilities. But the Mechanism in MeHg neurotoxicity is underunclear.Researches showed that cellular pathway play an important rolein neurodevelopment. So we assumed that PKC-RAS-MAPKs signalpathway maybe involved in MeHg neurotoxicity.Recently, the mechanisms about the activation PKC-Raf-MAPKspathway in neurotocxity were emphasized.Protein kinase C is a kind ofkinase that distributes widely in mammals cells, is a family that consistedof many kinds of isoenzymes, they have similar structures, but diferentbiochemical characteristics and physiological function.PKC is secondmessenger related to signal transduction from extracellular stimulation.MAPKs is a family of serine/threonine(Ser/Thr) kinases,which canbe activated by many factors, such as cytokines,hormones,neurotansm-itters and cell stress. MAPKs play a key role not only in cellularproliferation, but also in cellular differentiation and apoptosis. Inmammals,MAPK include 3 subtypes;ERK,JNK,p38. Signal pathways ofthese kinases are highly conserved.All these key kinases modulatecelular function through phosphorylated transcribe factors throughtransportation extracellular signal to nucleus. Diferent kinases act ondiferent substance to modulate diferent celular efects by relatedindependent signal pathway.ERK is one important family ofMAPKs,which can be activated by extracelular stimulation via G proteinreceptor, growth factor receptor and tyrosine protein kinase receptor andRaf-MEK-ERK series signal,then modulate transcribe factors and somekinases activites, promote protein synthesize, and cell proliferation.Apoptosis is relatedto neurotoxicity by prolonged low level exposure ofMeHg.Poly (ADP-ribose) polymerase (PARP) is involved in variouscellular functions, including DNA repair, the cell cycle and celldeath.While PARP activation could play a critical role in repairing braindamage, PARP inactivation caused by caspase 3-cleavage may also beimportant for apoptotic execution.PKC、MAPK and PARP are intracellularinteractional signal transfer.The aim of the present study was to investigate the role ofPKC-RAS-MAPKs signal pathway in mechanism of MeHgneurodevelopmental toxicity.For this purpose, PC12 cells were preparedand treated by low level MeHg, the cellular survival was determined withMTT assay,and apoptosis of PC12 cells was detected with flow cytometry(FCM).The effects of MeHg on the expression of PARP and PKCαandthe phosphorylation of ERK and JNK in PC12 cells and hippocampalneurons were detected by immunohistochemistry and western blot.Theresults show that low level exposure of MeHg affected the differentiationand survival of PC12 cells,and apoptosis of PC12 cells was alsostimulated.0.010-0.015μM MeHg increased the expression of PKCα andphosphorylation of ERK,however the expression of PKC α andphosphorylation of ERK were decreased and the expression of PARP andphosphorylation of JNK were increased by 0.020-0.030 μ M MeHg.Conclusion: The differentiation and survival can be affected by low levelexposure of MMC.The expression of PKCαand phosphorylation of ERKwere related to the alteration of PC12 cells’ differentiation.Thephosphorylation of ERK in PC12 cells and hippocampal neurons by MeHgwas regulated by PKC-Raf-ERK pthway. The expression of PKCα,PARPand the phosphorylation of ERK,JNK in mice hippocampal neurons werealtered by low level exposure of MMC,and the learning and memory abilityof mice was also decreased with the alteration in ultrastructure ofhippocampal neurons.The present study would be the roretical basis for theunderstanding in mechanism of neurotoxicity by MeHg.更多还原