砷诱导皮肤角化紊乱中Nrf2-ARE信号通路的作用及差异蛋白研究

发布时间：2018-06-02 20:47

本文选题：砷 + 角化　；参考：《新疆医科大学》2015年博士论文

【摘要】：目的:本研究通过体内、体外实验对家兔及人角质形成细胞染砷,了解砷在皮肤角质形成细胞和皮肤组织中的代谢规律及内暴露剂量,观察砷对皮肤角质形成细胞和皮肤组织的影响,探讨Nrf2-ARE信号通路在砷诱导皮肤角化紊乱中的作用及砷氧化损伤的皮肤毒性机制;通过对砷诱导皮肤角化紊乱差异蛋白的研究,在蛋白质分子水平探讨砷致皮肤角化紊乱的机制并寻找敏感标记物。为砷性皮肤损伤的防治提供新的思路及科学依据。方法:1)采用MTT还原法检测HacaT细胞生长情况,确定亚砷酸钠的LC50及染毒浓度,染毒浓度分别为24h LC50的1/50、1/20、1/10,即1.30μmol/L、3.25μmol/L、和6.50μmol/L;观察时间为24h、48h、72h;2)流式细胞仪检测HacaT细胞的凋亡情况;3)将30只健康成年清洁级雄性家兔随机分为5组,每组6只,分别为对照组(去离子水)及亚砷酸钠染毒组。采用自由饮水方式连续染毒12周。染毒剂量分别为LD50的1/100、1/50、1/20、1/10,即L:0.13mg/千克体重(kg.w)、M:0.26mg/(kg.w)、MH:0.65mg/(kg.w)和H:1.30mg/(kg.w);4)染毒12周结束后,家兔背部同一部位皮肤取样,电镜下观察皮肤细胞及组织的形态学改变;5)采集家兔处死前24h尿样,并取家兔肝脏组织,采用高效液相色谱-氢化物发生原子荧光光谱(HPLC-HGAFS)法检测家兔尿、肝脏、皮肤的iAsⅢ、iAsⅤ、一甲基胂酸(Monomethylarsonic acid,MMA)和二甲基胂酸(Dimethylarsinic acid,DMA)含量;6)通过实时荧光定量PCR(Real-time polymerase chain reaction,RT-PCR)检测染砷HacaT细胞和家兔皮肤Nrf2 mRNA的表达水平;7)通过相应试剂盒方法检测染砷家兔皮肤CAT、8-OHdG、MPO、GSH-Px、GST、SOD和MDA及HO-1含量或活力,总蛋白采用BCA法检测;8)用双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)筛选砷诱导角化紊乱家兔皮肤的差异蛋白点,胶内酶切后,利用基质辅助激光解析电离飞行时间质谱(Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry,MALDI-TOF-MS)结合NCBI(nr)非冗余数据库检索对差异蛋白进行鉴定;9)用RT-PCR检测HacaT细胞CK1、CK10和家兔皮肤CK1、CK10、CK2及蛋白质二硫键异构酶(protein disulfide isomerase,pdi)mrna的表达水平。结果:1)1.30μmol/l亚砷酸钠染毒48h能显著促进hacat细胞增殖;3.25μmol/l亚砷酸钠染毒96h、6.25μmol/l亚砷酸钠染毒72h显著抑制hacat细胞增殖(p均0.05);2)1.30μmol/l亚砷酸钠能抑制hacat细胞凋亡,24h时凋亡率最低;3.25μmol/l、6.25μmol/l亚砷酸钠染毒48h、72h可使hacat细胞凋亡率逐渐升高(p均0.05);3)电镜下可见,和对照组相比各染毒组家兔皮肤细胞形态和表皮尤其是皮肤角质层结构发生了改变。0.13mg/(kg.w)砷染毒12周即可出现角化紊乱,随砷染毒剂量的升高,家兔皮肤细胞形态改变及角化紊乱程度加重;4)染毒12周后家兔尿中总砷、iasⅢ及dma含量水平随砷染毒剂量的升高呈逐渐升高的趋势,与对照组比较,h砷染毒剂量组总砷、iasⅢ及dma含量水平升高(p均0.05)。iasⅤ含量水平无明显变化趋势。mma含量水平随砷染毒剂量升高呈现逐渐升高的趋势,与对照组比较,mh及h砷染毒剂量组其含量水平升高(p0.05)。与对照组比较,各砷染毒剂量组pmi值均升高(p0.05)。与对照组比较,除l砷染毒剂量组外,其余砷染毒剂量组smi值均升高(p0.05);各染毒组家兔皮肤总砷及dma含量高于对照组,且随染毒剂量升高而升高(p0.05);各形态砷中,dma含量最高。iasⅢ含量除mh组外,各染毒组高于对照组;iasⅤ含量各组均高于对照组,mma含量l组和h低于对照组,mh组高于对照组(p均0.05)。兔肝脏中总砷含量水平随砷染毒剂量的升高呈逐渐升高的趋势,与对照组比较,m、mh、h砷染毒剂量组总砷含量水平升高(p0.05)。肝脏中iasⅢ含量在各组间没有统计学差异;肝脏中iasⅤ含量m组与其他组相比升高(p0.05)。肝脏中mma含量在各组间没有统计学差异;肝脏中dma含量在各组中不同,与对照组相比,mh和h染砷剂量组其含量升高(p0.05)。家兔皮肤和尿中砷以dma形态为主,肝脏中各种形态砷分布未发现规律;随着砷染毒剂量升高,皮肤dma所占比例有升高的趋势。皮肤和尿的总砷和dma含量与肝脏总砷和dma含量呈正相关;5)亚砷酸钠染毒能使hacat细胞nrf2mrna表达发生改变,1.30μmol/l亚砷酸钠染毒使mrna表达上调,随着染毒时间延长、染毒浓度增加使mrna表达逐渐下调(p0.05)。砷染毒48h后hacat细胞nrf2mrna表达水平与细胞活性呈正相关关系;l、m组家兔皮肤nrf2mrna的表达上调;mh、h组皮肤nrf2mrna的表达下调,其中l、h组与对照组相比差异有统计学意义(p0.05)。6)染砷家兔皮肤8-ohdg在h组,mda在mh和h组比对照升高(p0.05);cat和ho-1在m、mh和h组,gsh-px在h组比对照降低(p0.05);gst在h组比对照升高(p0.05);sod和mpo在各组无明显差异。染砷家兔皮肤nrf2mrna表达与ho-1、gsh-px水平呈正相关关系,与mda、8-ohdg水平呈负相关关系;7)2-de电泳蛋白斑点清晰可见,各组蛋白斑点数在100-200之间,其中对照组蛋白斑点数为119,而其余4组蛋白斑点均为129个并全部匹配。对照组和其余各组的蛋白斑点匹配率为92.24%。与对照相比差异表达的蛋白点有45个,鉴定出20种差异蛋白。其中细胞角蛋白Ⅱ型蛋白、GST-P和PDI可能与砷的皮肤毒性有关;8)细胞角蛋白Ⅱ型蛋白在各砷染毒组家兔皮肤表达,而在对照组中无表达;HacaT染砷后CK1、CK10 mRNA表达发生改变,1.30μmol/L亚砷酸钠染毒能使mRNA表达上调,随着染毒时间延长、染毒浓度增加mRNA表达逐渐下调(P0.05);染砷家兔皮肤CK1、CK2、CK10 mRNA在L组表达上调,H组表达下降至正常或下调(P0.05)。染砷家兔皮肤PDI蛋白表达量随染砷剂量升高而先下调后上调;PDI mRNA在MH和H组,随染砷剂量升高表达量上升(P0.05)。结论:亚砷酸钠可在动物角质形成细胞和皮肤组织中发生生物转化并形成一系列代谢产物,随染毒剂量增加,皮肤砷负荷增加,可致皮肤氧化损伤,并引起角质形成细胞和皮肤组织Nrf2表达变化,并随染毒时间和浓度变化呈双向性;砷通过调控Nrf2水平继而影响皮肤角质形成细胞的增殖和凋亡及皮肤组织Nrf2-ARE信号通道下游酶的改变,皮肤氧化-抗氧化平衡被破坏,出现角化紊乱等形态学改变。砷代谢及Nrf2-ARE信号通路在砷致皮肤角化紊乱中发挥重要作用。差异蛋白研究发现PDI和CK1、CK2、CK10在砷诱导的皮肤角化紊乱中发挥作用;而细胞角蛋白异常是反应砷诱导皮肤角化紊乱较敏感的指标。
[Abstract]:Objective: To investigate the effects of arsenic on keratinocytes and skin tissues in skin keratinocytes and skin tissues, the effects of arsenic on keratinocytes and skin tissue of skin were observed and the role of Nrf2-ARE signaling pathway in the derangement of skin induced by arsenic was investigated. And the skin toxicity mechanism of arsenic oxidative damage; through the study of arsenic induced skin keratosis differential protein, the mechanism of arsenic induced dermatosis and sensitive markers were explored at the protein molecular level. New ideas and scientific basis for the prevention and control of arsenic skin injury were provided. Method: 1) the MTT reduction method was used to detect HacaT cell growth. For a long time, the LC50 and the concentration of sodium arsenite were determined and the concentration of the poisoned concentration was 24h LC50 1/50,1/20,1/10, 1.30 mu mol/L, 3.25 mu mol/L, and 6.50 mu mol/L, and the observation time was 24h, 48h, 72h; 2) flow cytometry to detect the apoptosis of HacaT cells; 3) 30 healthy adult clean grade male rabbits were randomly divided into 5 groups, 6 in each group, respectively The control group (deionized water) and sodium arsenite dyed group were treated with free drinking water for 12 weeks. The dose of 1/100,1/50,1/20,1/10, L:0.13mg/ kg body weight (kg.w), M:0.26mg/ (kg.w), MH:0.65mg/ (kg.w) and H:1.30mg/ (kg.w); 4) were taken at the end of the same part of the rabbit's back, and the skin was observed under the electron microscope, and the skin was observed under electron microscope. The morphologic changes of skin and tissue; 5) collect the 24h urine before death in rabbits and take the rabbit liver tissue, and use high performance liquid chromatography - hydride generation atomic fluorescence spectrometry (HPLC-HGAFS) to detect the urine, liver, iAs III, iAs V, Monomethylarsonic acid, MMA, and two methyl arsine (Dimethylarsinic acid, DM). A) content; 6) the expression level of arsenic HacaT cells and rabbit Nrf2 mRNA was detected by real-time fluorescent quantitative PCR (Real-time polymerase chain reaction, RT-PCR); 7) the arsenic rabbit skin was detected by the corresponding kit method. Two-dimensional gel electrophoresis (2-DE) was used to screen the differential protein points of the deranged rabbit skin with arsenic induced diagonalization. After the gel enzyme was cut, the matrix assisted laser analytical ionization time of flight mass spectrometry (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) was combined with non redundant data. 9) the expression level of HacaT cells CK1, CK10 and rabbit skin CK1, CK10, CK2 and protein two sulfur bond isomerase (protein disulfide isomerase, PDI) mRNA were detected by RT-PCR. Results: 1) 1.30 micron sodium arsenite could significantly promote cell proliferation; 3.25 Mu sodium arsenite was exposed to 6.25 mu. /l sodium arsenite exposure significantly inhibited the proliferation of HaCaT cells (P 0.05); 2) 1.30 mu mol/l sodium arsenite could inhibit apoptosis of HaCaT cells and the lowest apoptosis rate in 24h; 3.25 mu mol/l, 6.25 micron sodium arsenite infected 48h, 72h could increase the apoptosis rate of HaCaT cells (0.05); 3) under electron microscope, compared with the control group, the skin of each infected group was thin skin. The cell morphology and epidermis, especially the skin cuticle structure changed.0.13mg/ (kg.w) arsenic exposure for 12 weeks, can appear keratinization disorder. With the increase of arsenic exposure dose, the skin cell morphology change and the degree of keratinization disorder increased; 4) the total arsenic in urine in the rabbit after 12 weeks of exposure, the level of IAS III and DMA increased gradually with the increase of arsenic exposure dose. The rising trend, compared with the control group, the total arsenic, IAS III and DMA levels in the H arsenic exposure group increased (P 0.05), the level of.Ias V was not obviously changed, and the level of.Mma content increased gradually with the increase of arsenic exposure dose. Compared with the control group, the content level of MH and H arsenic poisoning group increased (P0.05). Compared with the control group, the content level of the arsenic Dyeing Group was higher than the control group. The PMI value of each arsenic exposure group increased (P0.05). Compared with the control group, the SMI value of the remaining arsenic exposure dose group increased (P0.05) except for the L arsenic exposure dose group, and the total arsenic and DMA content in the skin of the infected rabbits was higher than that in the control group, and increased with the increase of the dose (P0.05), and the highest.Ias III content of DMA content in each form of arsenic, except for MH group, was all dyed. The content of IAS V in each group was higher than that in the control group. The content of MMA content in group L and h was lower than that of the control group, and the MH group was higher than the control group (P 0.05). The level of total arsenic in the liver of the rabbit increased gradually with the increase of arsenic exposure dose. Compared with the control group, the level of total arsenic in the group of M, MH and H arsenic poisoning was increased (P0.05). The IAS III contained in the liver. There was no statistical difference between the groups. The content of IAS V in the liver was higher than that in the other groups (P0.05). There was no statistical difference in the content of MMA in the liver. The content of DMA in the liver was different in each group. Compared with the control group, the content of MH and h increased (P0.05). The arsenic in the skin and urine of the rabbit was mainly in the form of DMA, and in the liver, the liver was mainly in the liver and in the liver. The distribution of arsenic in various forms was not found. With the increase of arsenic exposure dose, the proportion of DMA in the skin increased. The total arsenic and DMA content in the skin and urine were positively correlated with the total arsenic and DMA content in the liver. 5) sodium arsenite can make the expression of nrf2mrna in HaCaT cells change, and the expression of mRNA is up to up with 1.30 mol/l sodium arsenite, with the increase of mRNA expression. The expression of mRNA expression gradually decreased (P0.05). The expression level of nrf2mrna in HaCaT cells was positively correlated with the activity of nrf2mrna after arsenic exposure to 48h; L, the expression of nrf2mrna in the skin of the rabbit group was up regulated, and the expression of nrf2mrna in the H group was down down, and the L, there was a significant difference between the H group and the control group. Rabbit skin 8-OHdG in group H, MDA in group MH and H (P0.05), cat and HO-1 in M, MH and H group, GSH-Px in the group compared with the control, and there is no significant difference in each group. 7) 2-DE electrophoresis protein spots were clearly visible, the number of spots in each group was 100-200, of which the number of spots in the control group was 119, and the other 4 groups of protein spots were all 129 and all matched. The matching rate of the protein spots in the control group and the rest of the other groups was 45, and 20 kinds of differential eggs were identified. Cytokeratin type II protein, GST-P and PDI may be related to the skin toxicity of arsenic; 8) cytokeratin type II protein is expressed in the skin of the rabbits exposed to arsenic, but no expression in the control group; the expression of CK1, CK10 mRNA in HacaT after arsenic contamination is changed, and the expression of mRNA in 1.30 Mu sodium arsenate can increase the expression of mRNA and as the time of exposure prolongs. The expression of mRNA, CK1, CK2, CK10 mRNA in the L group was up regulated and the expression of H group decreased to normal or down (P0.05). The expression of PDI protein in the skin of arsenic infected rabbits was down regulated with the increase of arsenic dose, and PDI mRNA was in the group and increased with the increase of arsenic dose. Sodium arsenite can produce biotransformation and form a series of metabolites in the keratinocytes and skin tissues of animals. As the dose increases, the arsenic load of skin increases, the skin oxidative damage can be induced, and the expression of Nrf2 in keratinocytes and skin tissues is changed, and the change of arsenic in the skin is bidirectional with the change of time and concentration, and arsenic is regulated by N The RF2 level affects the proliferation and apoptosis of skin keratinocytes and the changes in the downstream enzymes of the skin tissue Nrf2-ARE signaling pathway, the oxidative balance of the skin is destroyed and the morphological changes of keratinization disorder occur. Arsenic metabolism and Nrf2-ARE signaling pathway play an important role in arsenic induced derangement in the skin. Differential protein studies found PD I and CK1, CK2 and CK10 play a role in arsenic induced keratinization disorder, and cytokeratin abnormalities are sensitive indicators for arsenic induced keratinization disorders.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R135.1

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