【作者】 张江林；

【导师】 谢红付； 陈翔；

【作者基本信息】 中南大学 ， 皮肤病与性病学， 2008， 博士

【摘要】 流行病学研究表明低浓度砷剂的暴露与皮肤癌等各种肿瘤的发生有关。砷剂虽然被公认为是人类的致癌剂之一,但是其致癌作用机制仍存在争论。有的学者认为砷剂可单独引起肿瘤的发生,而有的学者认为砷剂只是协同其它致癌物导致正常细胞的恶性转化。p53基因是已知的抑癌基因之一,其介导的信号传导途径在细胞的增殖分化、应激和癌变等生命过程中起着极其重要的作用,被称为“基因组卫士”。鼠双微粒体2(murine double minute2,MDM2)基因是p53的下游基因之一,p53可激活MDM2转录,MDM2反过来又抑制p53的功能,二者形成自动调节反馈环,以保持正常情况下p53处于低水平状态。正确的亚细胞定位对于p53的功能活性十分重要,p53被激活后进入胞核内,激活其靶基因的转录,之后回到胞浆中被降解。MDM2由于具有核输出序列(NES)和核定位序列(NLS)而具有核浆穿梭能力,NES使MDM2由胞核到胞浆而NLS使之由胞浆到胞核。MDM2蛋白可把p53/MDM2复合物直接运出细胞核,MDM2的核浆穿梭能力对其抑制p53的功能活性非常重要。本课题研究中,我们以hTERT永生化的皮肤角质形成细胞为研究对象,探讨了砷剂对皮肤角质形成细胞p53功能的影响及MDM2在其中所起的作用机制,为阐明砷剂引起皮肤癌的机理提供新的实验依据。第一章低浓度砷剂经由上调MDM2介导p53胞浆分布所致功能失活目的:探讨低浓度砷剂对人皮肤角质形成细胞中MDM2蛋白的影响及其与p53的亚细胞分布状态和功能的关系。方法:以hTERT永生化的皮肤角质形成细胞为研究对象。采用蛋白印迹法(Western blotting)研究0.5μmol/L、1μmol/L和2μmol/L亚砷酸钠处理12小时和24小时后MDM2、p53和丝氨酸15位磷酸化的p53蛋白的表达水平,并且与5-Fu和紫外线处理作比较。采用细胞免疫荧光染色法研究砷剂处理前后p53蛋白的亚细胞定位及细霉素B和Nutlin-3预处理对其影响。结果:低浓度砷剂上调了角质形成细胞中MDM2蛋白的表达,但对p53蛋白表达无明显影响。随着低浓度砷剂剂量的逐渐增加,MDM2蛋白的表达水平亦逐渐增加;随着砷剂作用时间的逐渐增加,MDM2蛋白的表达水平也逐渐增强。低浓度砷剂处理后角质形成细胞p53蛋白主要分布在胞浆。细霉素B和Nutlin-3预处理可以阻断砷剂诱导的p53胞浆分布。先用低浓度砷剂处理角质形成细胞后再加5-Fu刺激,此时5-Fu激活p53功能受到明显的抑制。结论:低浓度砷剂呈剂量和时间依赖性上调角质形成细胞中MDM2蛋白的表达;低浓度砷剂可经由上调MDM2蛋白表达介导p53的胞浆分布;低浓度砷剂诱导的p53胞浆分布导致p53功能性失活。第二章低浓度砷剂上调MDM2的机制探讨目的:探讨低浓度砷剂上调MDM2表达的作用机制。方法:构建pGL3-MDM2基因启动子报告基因表达载体,采用荧光素酶报告基因分析方法观察亚砷酸钠对MDM2基因P1、P2启动子转录活性的影响。应用1μmol/L和2μmol/L亚砷酸钠处理皮肤角质形成细胞24h后采用逆转录-聚合酶链反应(RT-PCR)方法检测MDM2 mRNA的表达水平。应用PD98059、SB203580和LY294002等特异性信号转导通路抑制剂预处理后再观察砷剂对角质形成细胞MDM2表达的影响。结果:低浓度砷剂可显著诱导MDM2基因P1启动子的荧光素酶活性(P＜0.05)。随着低浓度砷剂的剂量逐渐增加,MDM2 mRNA的表达水平逐渐增高。PD98059预处理可完全阻断砷剂上调角质形成细胞中MDM2的表达,而SB203580和LY294002预处理对砷剂诱导的MDM2表达上调无明显影响。结论:通过MAPK/ERK信号转导通路,低浓度砷剂激活MDM2基因P1启动子的转录活性,从而诱导MDM2的表达。第三章低浓度砷剂通过功能性失活p53发挥辅助致癌作用目的:探讨低浓度砷剂对紫外线引起角质形成细胞凋亡的影响及其与砷性皮肤癌作用机制的关系。方法:应用0.5μmol/L和1μmol/L亚砷酸钠处理角质形成细胞24h后,再用40mJ/cm~2紫外线照射。采用流式细胞术和Hoechst33258胞核染色检测砷剂和UV引起的细胞凋亡。结果:0.5μmol/L和1μmol/L砷剂处理角质形成细胞后凋亡率分别为1.2%和1.5%,与正常对照组1.5%无显著性差异(P＞0.05)。而先经过0.5μmol/L和1μmol/L砷剂预处理后的角质形成细胞再照射紫外线,此时凋亡率分别为48.8%和39.9%,与未经砷剂预处理直接照射UV组凋亡率64.7%相比有显著性差异(P＜0.05)。表明砷剂预处理可导致角质形成细胞对UV的凋亡抗性。Hoechst33258核染色结果与之相一致。结论:低浓度砷剂暴露可损害皮肤角质形成细胞对紫外线引起的凋亡反应;低浓度砷剂可通过功能性失活p53从而发挥其辅助致癌作用。更多还原

【Abstract】 It has been proved by epidemiology that exposure of low concentration arsenic is associated with risk of cancers such as skin cancer.Although arsenic carcinogenesis is well documented,the mechanism of action is still debated.Some scientists intsist that arsenic can induce carcinogenesis by itself.Others hold that arsenic is only a co-carcinogen.P53 gene is one of the well-known antioncogenes.The signal trasnduction pathway mediated by p53 play the most importmant role in the cellular proliferation,differentiation,stress and carcinogenesis.P53 gene is named as "guardian of genome".MDM2（murine double minute 2, MDM2）gene is one of the downstream genes of p53.p53 can stimulate the transcription of MDM2 gene while MDM2 can inhibit the function of p53.So p53 and MDM2 form an autoregulated feed-back loop which keep p53 at low activity under normal condition.Accurate sub-cellular location is very important to p53 function. After activated,p53 go to nucleus and activate the transcription of target genes.Then p53 is degradated at cytoplasm.MDM2 protein can shuttle back and forth beween nucleus and cytoplasm due to its NES and NLS. NES can pull MDM2 protein from nucleus to cytoplasm while NLS can pull MDM2 protein from cytoplasm to nucleus.MDM2 protein can transport p53 /MDM2 complex between nucleus and cytoplasm.The ability of shuttling back and forth is very important for MDM2 to inhibit the function of p53.In our experment,the hTERT immortalized human keratinocytes were used to explore the effects of arsenic on p53 and MDM2.It will provide novel research findings to clarify arsenic carcinogenesis in the skin.The first chapter Low concentration arsenic functional inactivated p53 by cytoplasmic distribution through upregulation of MDM2Objective:To explore the effects of low concentration arsenic on MDM2 protein and association with p53 subcellular location and function in human keratinocytes.Methods:hTERT immmortalized human keratinocyte were used as model.Western blotting was used to explore the protein levels of MDM2, p53 and p53 phosphorylation at serine 15 site.After treatment of 0.5μmol/L,1μmol/L and 2μmol/L sodium arsenite for 12 hours or 24 hours.Immunofluorescence staining was used to observe the subcellular location of p53 protein before and after arsenic treatment and the effects of LMB and Nutlin-3.Results:The protein level of MDM2 was upregulated after low concentration arsenic administration while p53 protein has no obvious change.MDM2 protein increased steadily with the increase of dose and duration of arsenic treatment.Most of p53 protein located at cytoplasm in the keratinocyte after arsenic treatment.LMB and Nutlin-3 can block arsenic-induced p53 cytoplasmic distribution.Pretreatment of arsenic on keratinocyte can interfere with 5Fu-induced p53 activation.Conclusion:Low concentration arsenic upregulate MDM2 protein in a dose and time dependent manner.Low concentration arsenic induce p53 cytoplasmic distribution through upregulation of MDM2.Low dose arsenic-induced p53 cytoplasmic distribution functionally inactivated p53.The second chapter Mechanism of low concentration arsenic-induced upregulation of MDM2Objective:To explore the mechanism of action of upregulation of MDM2 protein after low dose arsenic treatment.Methods:The luciferase report gene system was used to observe the effects of sodium arsenite on MDM2 gene P₁、P₂ promoter.RT-PCR was used to explore the mRNA level of MDM2 after 1μmol/L and 2μmol/L sodium arsenite treatment in keratinocytes for 24 hours.Pretreatment of PD98059,SB203580 and LY294002 was used to interfere with the effects of sodium arsenite on MDM2 protein expression in keratinocytes.Results-The luciferase activity of MDM2 P₁ promoter was induced obviously after low concentration arsenic treatment（p＜0.05）.MDM2 mRNA level increased steadily with the increase of sodium arsenic. Pretreatment of PD98059 can totally block arsenic-induced MDM2 upregulation while SB203580 and LY294002 have no effect.Conclusion:Through MAPK/ERK pathway,low dose aresnic can induce the transciptional activity of P₁ promoter of MDM2 gene promoter, and then upregulate the expression of MDM2.The third chapter low concentration arsenic acts as a co-carcinogen by functional inactivation of p53Objective:To explore the effect of low dose arsenic on UV-induced apoptosis of keratinocyte and association with arsenic carcinogenesis in the skin.Methods:Flow cytometry was used to measure UV-induced apoptosis after 0.5μmol/L and 1μmol/L sodium arsenite treatment for 24 hours in keratinocytes.Results:The apoptotic rate of 0.5μmol/L and 1μmol/L sodium arsenite was 1.2%and 1.5%.There is no difference compared to 1.5%of control（P＞0.05）.After pretreated by 0.5μmol/L and 1μmol/L sodium arsenite,UV-induced apoptotic rate was 48.8%and 39.9%.There is significant difference compared to 64.7%of control（P＜0.05）.The results of Hoechst nuclei staining is the same as flow cytometry.Conclusion:Low dose arsenic exposure interfered with UVinduced apoptosis in keratinocytes.Low concentration of arsenic acts as a co-carcinogen by functional inactivation of p53.更多还原