【作者】 徐曼妮；

【导师】 李敏；

【作者基本信息】 第二军医大学 ， 营养与食品卫生学， 2013， 博士

【摘要】 铁是机体含量最丰富的金属元素之一，含铁蛋白在体内发挥重要的生理功能，参与氧的转运，细胞呼吸、物质代谢、转录调节和DNA修复。机体铁缺乏会产生缺铁性贫血和多种代谢障碍，然而过量的铁在体内能通过Fenton反应引起脂质过氧化，进一步损伤蛋白质和（或）核酸。机体铁负荷与癌症、冠心病、帕金森病、糖尿病等多种疾病的发生有关。肝脏是机体储存铁的主要器官，也是铁过量损伤的主要器官，有大量研究资料证实，肝铁过负荷会导致肝细胞坏死，纤维化和肝肿瘤，还能引起胰岛素抵抗、增加胆固醇的合成和沉积。因此探讨铁过负荷的原因，寻找防治的潜在靶点成为医学研究的热点。目前已证实HFE、HJV、TfR2等基因突变导致遗传性血色素沉着病，反复输血、膳食铁摄入过多等因素都可以引起机体铁过负荷，但是对于大多数疾病存在铁过负荷的原因尚未完全阐明。心理应激是指机体对外界有害物、威胁、挑战经认识评价后，知其将危害个人的生存和地位时，所产生的生理、心理和行为反应。现代生活中，人们在工作和生活中承受的负荷越来越大，精神紧张和心理压力已成为重要的应激源，心理因素对人体健康的影响正引起研究者广泛的关注。课题组前期的研究发现，心理应激可引起实验动物肝铁蓄积。其中部分原因可能是通过IL-6上调hepcidin及糖皮质激素对IRP1的调节，由于心理应激动物肝铁蓄积十分明显，是否还有其它的因素导致肝铁蓄积有待于进一步研究。为了深入分析心理应激导致肝铁蓄积的可能原因，本研究采用基因芯片技术筛选心理应激大鼠肝脏的差异表达基因，并根据基因表达趋势分析与肝铁负荷变化趋势相一致的基因，寻找与肝铁代谢变化有关的分子。结果发现在心理应激情况下，hepcidin、TfR1等已知的与铁代谢有关的分子发生了变化，还发现SFXN1的表达增强。SFXN1也称为三羧基携带蛋白TCC，是sideroflexin家族成员之一，定位于线粒体膜上，是真核生物的一种进化保守蛋白。有研究资料表明，SFXN1可能具有转运有利于铁利用的某种成分进入线粒体的功能，flexed-tail小鼠SFXN1发生了移码突变，表现出红细胞线粒体内铁沉积的特征。线粒体是细胞电子传递和能量代谢最为旺盛的细胞器，线粒体内铁代谢紊乱会严重地影响到整个细胞的铁代谢。因此，SFXN1是否对细胞铁代谢相关分子产生一定的影响，从而参与心理应激反应肝铁负荷的发生，我们进行了进一步实验研究。研究目的利用基因芯片筛选心理应激情况下大鼠肝脏的差异表达基因，并在此基础上寻找可能参与心理应激铁代谢异常的基因，为阐明心理应激导致肝铁负荷提供新的研究思路。研究方法一、心理应激大鼠血清铁和肝铁含量变化1、实验动物分组健康雄性SD大鼠48只（体重120±10g），购自中英合资上海西普尔-必凯实验动物有限公司。按体重随机分为对照组、1天、3天、7天心理应激组4组，每组8只，其余作为电击组。自由饮食，动物实验室条件为：温度24℃士1℃，湿度50％～60％，光照自然昼夜节律变化。2、制作心理应激模型大鼠心理应激模型制作采用Communication box system。该装置由透明丙烯酸板制成，所有小室的一半铺绝缘板（A室），另一半不铺绝缘板（B室）。B室的大鼠每天足底电击30分钟，电压为80V，大鼠被电击后尖叫、跳跃、排便；相邻A室的大鼠通过听觉、视觉和嗅觉产生心理应激。3、血清CORT、ACTH及NE测定按检测试剂盒说明测定血清CORT、ACTH及NE的浓度。4、测定肝铁、血清铁含量湿法消化肝组织，原子吸收分光光度计火焰法测定肝铁和血清铁含量。5、血清转铁蛋白饱和度的测定采用血清铁和血清总铁结合力试剂盒测定。二、心理应激大鼠肝脏SFXN1及铁代谢相关分子的变化1、实验动物分组及处理第一部分的实验动物，每组随机抽取三只大鼠，选择肝组织进行基因芯片分析。2、基因芯片分析采用Affymetrix公司的Rat Exon1.0STArray全转录组表达谱芯片，利用随机方差模型（RVM），以P﹤0.05和FDR﹤0.05为判断标准，进行差异基因筛选。用GO分类法查找与铁代谢有关的基因，并根据基因表达趋势分析与肝铁负荷变化趋势相一致的基因。3、大鼠肝脏hepcidin、TfR1、sfxn1mRNA表达的变化采用实时荧光定量PCR法测定大鼠肝脏内铁调素(hepcidin)、转铁蛋白受体1（transferrin receptor1，TfR1）、sideroflexin1（sfxn1）的mRNA表达变化。4、测定大鼠肝组织hepcidin、TfR1、sfxn1蛋白含量免疫印记法测定各组大鼠肝脏hepcidin、TfR1、sfxn1的蛋白含量。三、 SFXN1对肝细胞铁代谢主要调节分子的影响1、细胞培养HepG2肝细胞株，含10%胎牛血清的DMEM培养液37℃、5%CO2培养。2、Sfxn1siRNA转染细胞按说明书将sfxn1siRNA转染HepG2细胞后，培养箱中培养48h。3、肝细胞sfxn1、TfR1、hepcidin、IRP1、IRP2mRNA测定采用实时荧光定量PCR法测定HepG2细胞sfxn1、TfR1、hepcidin、IRP1、IRP2mRNA的表达水平。4、肝细胞sfxn1、TfR1、hepcidin、IRP1、IRP2的蛋白测定采用Western blot法测定HepG2细胞sfxn1、TfR1、hepcidin、IRP1、IRP2的蛋白含量。四、数据的统计与处理采用SPSS16.0统计软件包对实验数据进行统计分析。两组间比较采用两独立样本t-检验；多组间比较采用单因素方差分析，方差齐时各组样本均数间两两比较采用LSD-t检验，各实验组间与对照组比较采用Dunnett法。方差不齐时采用Dunnett’s C检验。实验数据以平均数±标准差（x±s）表示，p<0.05认为有统计学意义。结果一、心理应激大鼠血清铁和肝铁含量变化1、心理应激大鼠血清CORT、ACTH与NE含量变化心理应激大鼠血清CORT、ACTH、NE含量较对照组明显升高，说明心理应激模型成功制作。2、心理应激大鼠肝脏铁含量变化随着应激时间的延长，大鼠肝组织铁含量逐渐升高，7天心理应激组大鼠肝脏铁含量与对照组比较上升了53.02%（P<0.05）。1天和3天心理应激组大鼠肝铁含量与对照组比较无统计学意义（P>0.05）。3、心理应激大鼠血清铁含量变化1天和3天心理应激组大鼠血清铁含量与对照组比较无统计学意义（P>0.05）,与对照组比较，7天心理应激组大鼠血清铁含量下降了29.21%，差异有统计学意义（P<0.05）。4、心理应激大鼠血清转铁蛋白饱和度的变化与对照组比较，7天心理应激组大鼠血清转铁蛋白饱和度下降了32.2%（P<0.05）；1天和3天心理应激组大鼠血清转铁蛋白饱和度较对照组分别下降了7.87%、19.85%，但无统计学意义（P>0.05）。二、心理应激大鼠肝脏SFXN1及铁代谢相关分子的变化1、基因芯片筛选心理应激大鼠肝脏铁代谢相关差异基因在心理应激过程中，大鼠肝脏基因mRNA转录水平有明显差异的为2246个。对这些差异表达基因进行生物信息学分析，与铁代谢有关的差异基因有七个，分别为Hamp、Tfrc、sfxn1、GDF2、IREB2、NUBP1、NUBP2。sfxn1基因的表达趋势分析显示其变化趋势与肝铁沉积变化趋势一致。2、心理应激大鼠肝脏hepcidin、TfR1mRNA的表达变化7天心理应激后大鼠肝脏hepcidin mRNA的表达水平较对照、1天、3天心理应激组均有显著升高（P<0.01）；心理应激第七天TfR1mRNA的表达水平比对照、1天、3天心理应激组升高，并有统计学意义。3、心理应激大鼠肝脏hepcidin、TfR1的蛋白表达变化7天心理应激组hepcidin蛋白的表达水平较对照、1天、3天心理应激组均有显著升高（P<0.01）。TfR1蛋白在应激后逐渐升高，7天心理应激组TfR1蛋白的表达水平与对照组比较有统计学意义（P<0.01）。4、心理应激大鼠肝脏sfxn1的表达变化应激后sfxn1mRNA的水平逐渐升高，3天应激组与对照组、1天应激组比较有统计学意义（P<0.01）；7天应激组sfxn1mRNA的表达水平比对照、1天、3天心理应激组升高，并有统计学意义（P<0.01）。Sfxn1蛋白在应激后逐渐升高，7天心理应激组的表达水平较对照组升高66%（P<0.01）。三、SFXN1对肝细胞铁代谢主要调节分子的影响1、Sfxn1siRNA转染对HepG2细胞sfxn1mRNA表达的影响HepG2细胞转染sfxn1siRNA后sfxn1mRNA的表达量较未转染的对照组降低（P<0.05）。2、Sfxn1siRNA转染对HepG2细胞TfR1、 hepcidin mRNA表达的影响Sfxn1siRNA转染HepG2细胞48小时后，转染组TfR1mRNA的表达量较未转染组降低了78%（P<0.01）。Hepcidin mRNA的表达量与对照组比较无区别。3、Sfxn1siRNA转染对HepG2细胞IRP1、 IRP2mRNA表达的影响Sfxn1siRNA转染HepG2细胞48小时后，转染组IRP1mRNA的表达量较未转染组降低了80%（P<0.05）。转染组IRP2mRNA的表达量与对照组比较无差别。4、Sfxn1siRNA转染降低HepG2细胞sfxn1蛋白表达Sfxn1siRNA转染HepG2细胞后使sfxn1蛋白表达下降（P<0.01）。5、Sfxn1siRNA干扰对HepG2细胞TfR1、 hepcidin蛋白表达的影响Sfxn1siRNA转染HepG2细胞后使TfR1蛋白表达下降（P<0.01）。hepcidin的蛋白表达不受影响。6、Sfxn1siRNA干扰对HepG2细胞IRP1、 IRP2蛋白表达的影响Sfxn1siRNA转染HepG2细胞后使IRP1蛋白表达下降（P<0.01）。IRP2的表达未发生改变。结论1.连续7天心理应激可以引起大鼠肝铁蓄积。2.基因芯片发现心理应激过程中Hamp、Tfrc、sfxn1、GDF2、IREB2、NUBP1、NUBP2共7个与铁代谢相关的基因发生了改变。hepcidin、TfR1、sfxn1的mRNA和蛋白水平随着应激天数的增加不断上升。3. sfxn1能影响TfR1的表达综上所述，心理应激时可能通过sfxn1引起转铁蛋白受体1表达增加，肝细胞摄入铁增加，导致肝铁蓄积。更多还原

【Abstract】 BackgroundIron is the most abundant transition metal in the body that is mainly present inprotein-bound forms such as heme and non-heme proteins, playing a major role in electrontransfer and oxygen utilization. Iron deficiency and iron overload can occur and may haveserious clinical consequences. The most common disorder associated with iron depletion isiron deficiency anemia,which affects more than30%of the world’s population.At the otherend of the spectrum,iron overload can occur.This transition metal promotes free radicalgeneration through Fenton and/or Haber–Weiss reactions, thus triggering secondary chainreactions in the oxidative modification of lipids, proteins, and DNA in different organs. Soiron metabolism is tightly regulated.The liver is the major storage site for iron.But excessive iron deposition in liver willlead to further injury such as hepatocellular necrosis, inflammation, fibrosis and in somecases even to carcinoma. Hepatic iron overload could contribute to the development ofNAFLD or lipotoxicity.Epidemiological studies have shown that psychological stress may lead to a variety ofdiseases, such as hypertension, ulcer disease, cerebrovascular accident, myocardialinfarction, diabetes, cancer, etc.Our previous studies have shown that psychological stress can increase the liver irondeposition, but the exact mechanism is still not very clear. In this study, The technique ofmicroarray was used to analysis the liver gene expression profiles of psychological stressrat and to screen out iron related genes.We find out that psychological stress can increasesfxn1expression. Sfxn1also known as tricarboxylate carrier protein.It’s a member of thesideroflexin family. It’s located in the mitochondrial membrane. Sfxn1function might beinvolved in the transport of a component required for iron utilization into or out of themitochondria. But it is still unclear the exact role of the sfxn1. We study the sfxn1if it caneffect iron metabolism related molecules, in order to provide new ideas about thepsychological stress cause liver iron overload.ObjectiveIn this study, we use gene chip screen liver differentially expressed genes in PS rats.On this basis to find genes may be involved in iron metabolism of psychological stress, provide a new idea about the psychological stress cause liver iron overload. To clarify thepsychological stress how to lead to liver iron overload.Methods1. Effects of psychological stress on the rat serum iron and hepatic iron concentration1.1Experimental animals48male Sprague Dawley rat（s120±10g）were purchased from Shanghai-BK Ltd. Afteradaptation for5days, the rats were randomized into three groups: control group(n=8),psychological stress groups(futher divided into three subgroup as PS-1,PS-3,PS-7,with8rats in each subgroup),food-shocked group. Psychological stress rats were exposed tostress for30minutes every day. Animal were housed in a temperature-controlled room at24℃under a12-hour light/dark cycle. All animals used in this study received anutritionally-balanced rodent diet with “normal” iron level (35mg iron/kg diet) and waterad libitum. All animal procedures are carried out in accordance with establishedprocedures, Second Military Medical University Animal Care and Use Committee.1.2Build psychological stress model of SD ratsThe communication box was selected as the psychological stress apparatus. It dividedinto20compartments by transparent acrylic board with several small hole. The boardprevented each rat from physical contact but allowed them to receive cues such as visual,auditory and olfactory sensations from the neighboring animals. The compartments weredivided into two parts. Half of all small rooms spread insulation panels (A Room), and theother half do not shop insulated panels (B room). An electric generator with80voltageswas connected to the grid floor to generate electric foot shock.Rats in part B after theshock,exhibited a nociceptive stimulation-evoked response such as screaming,jumping,defecation. Rats in part A were not directly administered the foot electrical shock, butproduced psychological stress.1.3Measurement of serum ACTH、CORT and NE concentrationsSerum ACTH concentration was measured by the enzyme-linked immunoassays(ELISAs) kit; the CORT and NE levels were measured by the radioimmunofocus assays(RI) kit. 1.4Measurement of liver iron concentrationSpecimens were weighed and digested with concentrated nitric-perchloric acid mixture(4to1ratio). Iron estimated by atomic absorption spectrophotometry.1.5Determination of serum ironSerum iron concentration was determined in nonhemolyzed serum samples by atomicabsorption spectrophotometer.1.6Determination of serum Transferrin SaturationAccording to the manufacturer’s protocol,serum iron and total iron bindingcapacity(TIBC) were determined.TS(%)=serum iron/TIBC×100%2. Gene expression analysis of iron related genes in PS rat2.1Experimental animals and treatmentSame as in part1.2.2Gene expression profile analysisThe technique of cDNA microarray was used to screen out iron related genes onpsychological stress rats．Before and1，3，7day psychological stress，total liver RNA wereextracted and detected by Rat Exon1.0ST Array Gene Chip．We analysised the liver geneexpression profiles and then searched the iron related genes use gene ontology description.2.3Real time PCR analysis for hepcidin、TfR1、sfxn1mRNA expressionTotal RNA was extracted from liver tissue using TRIzol reagent and cDNA wasprepared. Real-Time PCR was performed for individual genes using SYBR green detectionsystem. The RT-PCR data were analyzed by standard curve method as described.2.4Western blot analysis for hepcidin、TfR1、sfxn1protein levelsLivers were homogenized with lysis buffer. Proteins were quantified using the kanjiprotein assay kit based on the Bradford method. Aliquots of protein were loaded on10%sodium dodecyl sulfate polyarylamid gel electrophoresis under reducing conditions andsubsequently transferred to a PVDF membrane. The blot were blocked and probed withantibodies.The intensity of the specific bands was detected and analyzed.To ensure evenloading of the samples,the same membrane was proed with rabbit anti rat GAPDH polyclonal antibody.3. The effect of sfxn1on hepatic cell iron-related molecules3.1Cell CultureHepG2(human liver hepatocellular carcinoma cell line) was purchased from TypeCulture Collection of the Chinese Academy of Sciences, Shanghai. HepG2cells weregrown at37°C with5%CO2in DMEM medium supplemented with10%heat-inactivatedfetal bovine serum (FBS),100U/ml penicillin and100ug/ml streptomycin.3.2Sfxn1siRNA transfectionHepG2cells were transfected with sfxn1siRNA as protocol described.3.3Determination of sfxn1、TfR1、hepcidin、IRP1、IRP2mRNA levels in HepG2cellsSame as in part2.3.3.4Determination of sfxn1、TfR1、hepcidin、IRP1、IRP2protein level in HepG2cellsExperimental methods see part2.4.4. Statistical analysisStatistical analyses were performed using SPSS software for Windows (version16.0)(SPSS, Inc., Chicago, IL, USA). Data were presented as mean±S.D. All the experimentaldatas were analyzed by independent sample T test method or one-way ANOVA.Aprobability value of P <0.05was taken to be statistically significant.Results1. Effects of psychological stress on the rat serum iron and hepatic iron concentration1.1Determination of the PS model establishmentAfter psychological stress exposure the levels of serum ACTH, corticosterone andNE were significantly higher than those in the control animals (P<0.05),indicating thePS model is establishment successfully.1.2Effect of psychological stress on liver iron concentrationsWe found that rat liver iron concentrations of7day PS group than the control groupincreased significantly (P <0.05), but1day、3day PS exposure groups no differencecompared with the control group. 1.3Effect of psychological stress on serum iron concentrationsCompared with the control group,1d-,3d-and7d psychological stress group serumiron decreased by11.32%,25.53%and29.21%respectivlely, the serum iron levels in7days PS exposure group significantly lower than the control group (P<0.05).1.4Effect of psychological stress on serum Transferrin SaturationCompared with control group,7days psychological stress group serum transferrinsaturation decreased32.2%(P<0.05),1and3days psychological stress group Serumtransferrin saturation fell7.87%,19.85%(P>0.05).2. Gene expression analysis of iron related genes in PS rat2.1Gene expression profile analysisIn the psychological stress rat liver, The significant difference genes number is2246.There are seven genes related with iron metabolism is changed.2.2PS exposure caused changes in hepcidin、TfR1mRNA levelsAfter seven days of psychological stress, rat liver hepcidin mRNA expression levelswere significantly increased（P<0.01), but compared with the control group,1day and3days PS exposure did not change (P>0.05). TfR1mRNA levels were gradully increased in1day,3days and7days PS groups, but7days PS exposure group changed significantly(P<0.01).2.3PS exposure changed hepcidin、TfR1protein expressionAfter7days PS exposure, rat liver hepcidin and TfR1protein levels were significantlyhigher than the control group(P<0.05)..2.4PS exposure increase sfxn1expression in rat liverSfxn1mRNA levels were increased in3days、7days PS exposure compared with thecontrol group (P<0.01). After7days PS exposure, Sfxn1protein levels weresignificantly higher than the control group (P<0.05).3. The effect of sfxn1on hepatic cell iron-related molecules3.1Effect of sfxn1siRNA on sfxn1expression levels in HepG2cellsThe mRNA expression of sfxn1was significantly decreased in siRNA transfection HepG2cell (P<0.05). Sfxn1protein level was significantly decreased in siRNAtransfection group (P<0.01).3.2Sfxn1interference decrease mRNA expression of TfR1、IRP1The TfR1、IRP1mRNA levels decreased in siRNA interference groups compared tocontrol group (P<0.05).But hepcidin、IRP2mRNA showed no difference in siRNAinterference group compared with control group (P>0.05).3.3Effects of sfxn1interference on TfR1、IRP1protein levelsThe TfR1、IRP1protein levels decreased in siRNA interference groups compared tocontrol group (P<0.05).Conclusions1.7days PS could cause iron accumulation in liver.2. The gene chip found Hamp, Tfrc, sfxn1, GDF2,IREB2, NUBP1, NUBP2genes relatediron homeostasis is changed in the process of psychological stress.3. Hepcidin,TfR1and sfxn1expression is rising with the increase in the number of stressdays.4. sfxn1can affect the expression of TfR1Taken together, we found psychological stress can increase transferrin receptor1expression may be by sfxn1,Result in liver cells intake of iron increased, made ironaccumulation in liver and even damage to liver.更多还原