心理应激大鼠肝脏SFXN1的表达变化及其对肝细胞铁代谢相关分子的影响
发布时间：2018-07-25 12:12
【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㩳0.05和FDR㩳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，p0.05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%（P0.05）。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（P0.05）。 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（P0.05）,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（P0.05）。 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%（P0.05）；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（P0.05）。 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（P0.01）；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（P0.01）。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（P0.01）。 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（P0.01）；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（P0.01）。 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%（P0.01）。 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（P0.05）。 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%（P0.01）。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%（P0.05）。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（P0.01）。 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（P0.01）。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（P0.01）。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。
[Abstract]:Iron is one of the most abundant metal elements in the body. Ferritin plays an important physiological function in the body. It participates in the transport of oxygen, cell respiration, substance metabolism, transcription regulation and DNA repair. Iron deficiency in the body produces iron deficiency anemia and a variety of metabolic disorders. However, excessive iron can cause lipid peroxidation in the body through Fenton reaction. Further damage to protein and / or nucleic acid. Iron load in the body is associated with a variety of diseases such as cancer, coronary heart disease, Parkinson's disease, and diabetes. The liver is the main organ for storing iron and is also the main organ of iron excess injury. A large number of research data have proved that liver iron overload will lead to necrosis of liver cells, fibrosis and liver tumors. It can cause insulin resistance and increase the synthesis and deposition of cholesterol. Therefore, it has become a hot topic in medical research to explore the causes of iron overload and to find potential targets for prevention and treatment. It has been proved that genetic mutations in HFE, HJV, TfR2 and other factors lead to the excessive negative factors of iron intake, such as repeated blood transfusion, and excessive dietary iron intake. However, the cause of iron overload in most diseases has not been fully elucidated.
Psychological stress refers to the physiological, psychological and behavioral responses of the body to the external harmful objects, threatening and challenging the individual's survival and status. In modern life, people have become more and more loaded in their work and life. Mental tension and psychological pressure have become an important source of stress and psychological factors in modern life. The effect on human health is causing widespread concern. Previous research in the group found that psychological stress could cause the accumulation of liver iron in experimental animals. Some of these may be due to the regulation of hepcidin and glucocorticoids on IRP1 through IL-6. There are other factors due to the obvious accumulation of iron in the liver of animals with psychological stress. The accumulation of liver iron remains to be further studied.
In order to analyze the possible causes of the accumulation of liver iron caused by psychological stress, the gene chip technique was used to screen the differentially expressed genes in the liver of psychological stress rats, and to find the molecules related to the changes of liver iron metabolism according to the analysis of the trend of the change of liver iron load. The results were found in psychological stress. In the case of hepcidin, TfR1 and other known molecules related to iron metabolism, the expression of SFXN1 is also known as the three carboxyl carrying protein TCC, which is one of the members of the sideroflexin family, located on the mitochondrial membrane, and is an evolutionary conservative protein of eukaryotes. Research data suggest that SFXN1 may have transport. It is beneficial to the function of iron utilization to enter the mitochondrial function. The flexed-tail mouse SFXN1 has a code shift mutation, showing the characteristics of iron deposition in the mitochondria of the erythrocytes. Mitochondria are the most powerful organelles of cell electron transfer and energy metabolism, and the iron metabolism disorder in mitochondria will seriously affect the iron metabolism of the whole cell. Therefore, S Whether FXN1 has a certain influence on the cell iron metabolism related molecules, and thus participate in the occurrence of liver iron load in the psychological stress response, we have carried out a further experimental study.
research objective
The gene chip is used to screen the differentially expressed genes in the rat liver under psychological stress, and on this basis, we find the genes that may participate in the abnormal iron metabolism in psychological stress, so as to provide a new way of studying the liver iron load caused by psychological stress.
research method
Changes in serum iron and liver iron contents in rats with psychological stress
1, group of experimental animals
48 healthy male SD rats (weight 120 + 10g) were purchased from the Sino British joint venture Shanghai saikole laboratory animal Co., Ltd., which were randomly divided into the control group, 1 days, 3 days, 7 days of psychological stress group 4 groups, 8 in each group, and the rest as the electric shock group. The free diet and animal laboratory items were: temperature 24, 1, 50% ~ 60%, light natural day The night rhythm changes.
2, making the psychological stress model
The rat model of psychological stress was made by Communication box system., which was made of transparent acrylic plate, half of the insulating board in all small rooms (room A), and the rats in the.B room of the other half of the non insulating board (B room) the electric shock of the foot was 30 minutes per day, the voltage was 80V, and the rats were screamed, jumped and defecate after the shock; rats in the adjacent A room were heard through hearing, depending on hearing. Sense and sense of smell produce psychological stress.
3, determination of serum CORT, ACTH and NE
The concentrations of CORT, ACTH and NE in serum were determined according to the test kit.
4, determination of liver iron and serum iron content
The liver tissue was digested by wet method and the iron and serum iron contents were determined by flame atomic absorption spectrophotometer.
5, determination of serum transferrin saturation
The serum iron and serum total iron binding capacity were determined by kit.
Two, the changes of SFXN1 and iron metabolism related molecules in the liver of rats with psychological stress.
1, group and treatment of experimental animals
In the first part of the experimental animals, three rats were randomly selected from each group, and liver tissues were selected for gene chip analysis.
2, gene chip analysis
Using the Rat Exon1.0STArray total transcriptional chip of Affymetrix company, using the random variance model (RVM), using P? 0.05 and FDR? 0.05 as the criterion, differential gene screening was carried out. The genes related to iron metabolism were found by GO taxonomy, and the gene expression trend was consistent with the change trend of liver iron load.
3, the expression of hepcidin, TfR1 and sfxn1mRNA in rat liver
The real time fluorescence quantitative PCR method was used to determine the changes in the expression of hepcidin, transferrin receptor 1 (transferrin receptor1, TfR1), and sideroflexin1 (sfxn1) in rat liver.
4, the contents of hepcidin, TfR1 and sfxn1 proteins in rat liver tissues were measured.
The contents of hepcidin, TfR1 and sfxn1 in liver of each group were measured by immuno imprinting.
Three, the effect of SFXN1 on the major regulatory molecules of iron metabolism in liver cells.
1, cell culture
HepG2 hepatocyte strain, DMEM culture medium containing 10% fetal bovine serum, was cultured at 37 C and 5%CO2.
2, Sfxn1siRNA transfected cells
Sfxn1siRNA was transfected into HepG2 cells according to instructions, and 48h. was cultured in incubator.
3, determination of hepatocyte sfxn1, TfR1, hepcidin, IRP1, IRP2mRNA
The expression levels of sfxn1, TfR1, hepcidin, IRP1 and IRP2mRNA in HepG2 cells were measured by real-time fluorescence quantitative PCR.
4, determination of protein sfxn1, TfR1, hepcidin, IRP1 and IRP2 in hepatocytes
The contents of sfxn1, TfR1, hepcidin, IRP1 and IRP2 in HepG2 cells were determined by Western blot.
Four, statistics and processing of data
SPSS16.0 statistical software package was used to analyze the experimental data. Two independent sample t- tests were used between the two groups; the multiple groups were compared by single factor analysis of variance, and 22 of the samples were compared with the LSD-t test, and the Dunnett method was compared with the control group. Dunnett 's was used when the variance was uneven. C test. The experimental data were expressed by mean + standard deviation (x + s), and P0.05 thought it was statistically significant.
Result
Changes in serum iron and liver iron contents in rats with psychological stress
1, the changes of serum CORT, ACTH and NE levels in rats with psychological stress.
The contents of CORT, ACTH and NE in psychological stress rats were significantly higher than those in the control group, indicating that the psychological stress model was successfully produced.
2, changes of iron content in the liver of rats with psychological stress
With the prolongation of stress time, the iron content of liver tissue in rats increased gradually. The iron content in liver of the rats in the psychological stress group increased by 53.02% (P0.05) and 3 days in the psychological stress group. The iron content of the rats in the psychological stress group had no significant difference between the control group and the control group on the 7 day (P0.05).
3, change of iron content in serum of psychological stress rats
There was no significant difference in serum iron content between the 1 days and 3 days of psychological stress group with the control group (P0.05). Compared with the control group, the serum iron content of the rats in the psychological stress group decreased by 29.21% in 7 days, and the difference was statistically significant (P0.05).
4, the change of serum transferrin saturation in rats with psychological stress.
Compared with the control group, the serum transferrin saturation of the rats in the 7 day psychological stress group decreased by 32.2% (P0.05), and the serum transferrin saturation of the rats in the psychological stress group was 7.87% and 19.85%, respectively, on 1 and 3 days, but there was no statistical significance (P0.05).
Two, the changes of SFXN1 and iron metabolism related molecules in the liver of rats with psychological stress.
1, gene chip screening of genes related to iron metabolism in rats with psychological stress.
In the process of psychological stress, there are 2246 significant differences in the transcriptional level of liver gene mRNA in rats. There are seven differentially differentially expressed genes related to iron metabolism for these differentially expressed genes, which are Hamp, Tfrc, sfxn1, GDF2, IREB2, NUBP1, and NUBP2.sfxn1 gene expression trend analysis show that the trend of the change and liver iron precipitation The trend of product change is consistent.
2, the expression of hepcidin and TfR1mRNA in the liver of rats with psychological stress.
After 7 days of psychological stress, the expression level of hepcidin mRNA in the rat liver was significantly higher than that in the control group, 1 days and 3 days in the psychological stress group (P0.01), and the expression level of TfR1mRNA in seventh days of psychological stress was higher than that of the control, 1 days, and 3 days of psychological stress group, and had statistical significance.
3, the expression of hepcidin and TfR1 protein in the liver of rats with psychological stress.
The expression level of hepcidin protein in the 7 day psychological stress group was significantly higher than that of the control group, 1 days, and the 3 days of psychological stress group increased significantly (P0.01).TfR1 protein increased gradually after stress. The expression level of TfR1 protein in the psychological stress group was statistically significant (P0.01) on the 7 day stress group.
4, changes in the expression of sfxn1 in the liver of rats with psychological stress
After stress, the level of sfxn1mRNA increased gradually. The 3 day stress group was significantly higher than the control group and the control group and the 1 day stress group (P0.01). The expression level of sfxn1mRNA in the 7 day stress group was higher than the control group, 1 days and 3 days of psychological stress group, and had statistical significance (P0.01).
Sfxn1 protein increased gradually after stress. On the 7 day, the expression level of psychological stress group was 66% higher than that of the control group (P0.01).
Three, the effect of SFXN1 on the major regulatory molecules of iron metabolism in liver cells.
1, the effect of Sfxn1siRNA transfection on the expression of sfxn1mRNA in HepG2 cells.
The expression of sfxn1mRNA in HepG2 cells transfected with sfxn1siRNA was lower than that in untransfected control group (P0.05).
2, the effect of Sfxn1siRNA transfection on the expression of TfR1 and hepcidin mRNA in HepG2 cells.
After Sfxn1siRNA transfection of HepG2 cells for 48 hours, the expression of TfR1mRNA in the transfected group decreased by 78% (P0.01).Hepcidin mRNA than that in the control group, and there was no difference compared with the control group.
3, the effect of Sfxn1siRNA transfection on the expression of IRP1 and IRP2mRNA in HepG2 cells.
After Sfxn1siRNA transfection of HepG2 cells for 48 hours, the expression of IRP1mRNA in the transfected group was 80% lower than that in the untransfected group (P0.05). There was no difference in the expression of IRP2mRNA in the transfected group compared with the control group.
4, Sfxn1siRNA transfection reduced the expression of sfxn1 protein in HepG2 cells.
After transfection of HepG2 cells with Sfxn1siRNA, the expression of sfxn1 protein decreased (P0.01).
5, the effect of Sfxn1siRNA interference on the expression of TfR1 and hepcidin protein in HepG2 cells.
Sfxn1siRNA transfected HepG2 cells reduced the expression of TfR1 protein (P0.01) and the protein expression of.Hepcidin was not affected.
6, the effect of Sfxn1siRNA interference on the expression of IRP1 and IRP2 protein in HepG2 cells.
After transfection of HepG2 cells with Sfxn1siRNA, the expression of IRP1 protein decreased (P0.01) and.IRP2 expression did not change.
conclusion
1. psychological stress for 7 consecutive days can cause liver iron accumulation in rats.
2. gene chip found that Hamp, Tfrc, sfxn1, GDF2, IREB2, NUBP1, NUBP2 in psychological stress changed.Hepcidin, TfR1, sfxn1 mRNA and protein levels increased with the increase of the number of stress days.
3. sfxn1 can affect the expression of TfR1
In summary, psychological stress may increase the expression of transferrin receptor 1 through sfxn1, and increase iron intake in liver cells, resulting in liver iron accumulation.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R151.2
本文编号：2143785
[Abstract]:Iron is one of the most abundant metal elements in the body. Ferritin plays an important physiological function in the body. It participates in the transport of oxygen, cell respiration, substance metabolism, transcription regulation and DNA repair. Iron deficiency in the body produces iron deficiency anemia and a variety of metabolic disorders. However, excessive iron can cause lipid peroxidation in the body through Fenton reaction. Further damage to protein and / or nucleic acid. Iron load in the body is associated with a variety of diseases such as cancer, coronary heart disease, Parkinson's disease, and diabetes. The liver is the main organ for storing iron and is also the main organ of iron excess injury. A large number of research data have proved that liver iron overload will lead to necrosis of liver cells, fibrosis and liver tumors. It can cause insulin resistance and increase the synthesis and deposition of cholesterol. Therefore, it has become a hot topic in medical research to explore the causes of iron overload and to find potential targets for prevention and treatment. It has been proved that genetic mutations in HFE, HJV, TfR2 and other factors lead to the excessive negative factors of iron intake, such as repeated blood transfusion, and excessive dietary iron intake. However, the cause of iron overload in most diseases has not been fully elucidated.
Psychological stress refers to the physiological, psychological and behavioral responses of the body to the external harmful objects, threatening and challenging the individual's survival and status. In modern life, people have become more and more loaded in their work and life. Mental tension and psychological pressure have become an important source of stress and psychological factors in modern life. The effect on human health is causing widespread concern. Previous research in the group found that psychological stress could cause the accumulation of liver iron in experimental animals. Some of these may be due to the regulation of hepcidin and glucocorticoids on IRP1 through IL-6. There are other factors due to the obvious accumulation of iron in the liver of animals with psychological stress. The accumulation of liver iron remains to be further studied.
In order to analyze the possible causes of the accumulation of liver iron caused by psychological stress, the gene chip technique was used to screen the differentially expressed genes in the liver of psychological stress rats, and to find the molecules related to the changes of liver iron metabolism according to the analysis of the trend of the change of liver iron load. The results were found in psychological stress. In the case of hepcidin, TfR1 and other known molecules related to iron metabolism, the expression of SFXN1 is also known as the three carboxyl carrying protein TCC, which is one of the members of the sideroflexin family, located on the mitochondrial membrane, and is an evolutionary conservative protein of eukaryotes. Research data suggest that SFXN1 may have transport. It is beneficial to the function of iron utilization to enter the mitochondrial function. The flexed-tail mouse SFXN1 has a code shift mutation, showing the characteristics of iron deposition in the mitochondria of the erythrocytes. Mitochondria are the most powerful organelles of cell electron transfer and energy metabolism, and the iron metabolism disorder in mitochondria will seriously affect the iron metabolism of the whole cell. Therefore, S Whether FXN1 has a certain influence on the cell iron metabolism related molecules, and thus participate in the occurrence of liver iron load in the psychological stress response, we have carried out a further experimental study.
research objective
The gene chip is used to screen the differentially expressed genes in the rat liver under psychological stress, and on this basis, we find the genes that may participate in the abnormal iron metabolism in psychological stress, so as to provide a new way of studying the liver iron load caused by psychological stress.
research method
Changes in serum iron and liver iron contents in rats with psychological stress
1, group of experimental animals
48 healthy male SD rats (weight 120 + 10g) were purchased from the Sino British joint venture Shanghai saikole laboratory animal Co., Ltd., which were randomly divided into the control group, 1 days, 3 days, 7 days of psychological stress group 4 groups, 8 in each group, and the rest as the electric shock group. The free diet and animal laboratory items were: temperature 24, 1, 50% ~ 60%, light natural day The night rhythm changes.
2, making the psychological stress model
The rat model of psychological stress was made by Communication box system., which was made of transparent acrylic plate, half of the insulating board in all small rooms (room A), and the rats in the.B room of the other half of the non insulating board (B room) the electric shock of the foot was 30 minutes per day, the voltage was 80V, and the rats were screamed, jumped and defecate after the shock; rats in the adjacent A room were heard through hearing, depending on hearing. Sense and sense of smell produce psychological stress.
3, determination of serum CORT, ACTH and NE
The concentrations of CORT, ACTH and NE in serum were determined according to the test kit.
4, determination of liver iron and serum iron content
The liver tissue was digested by wet method and the iron and serum iron contents were determined by flame atomic absorption spectrophotometer.
5, determination of serum transferrin saturation
The serum iron and serum total iron binding capacity were determined by kit.
Two, the changes of SFXN1 and iron metabolism related molecules in the liver of rats with psychological stress.
1, group and treatment of experimental animals
In the first part of the experimental animals, three rats were randomly selected from each group, and liver tissues were selected for gene chip analysis.
2, gene chip analysis
Using the Rat Exon1.0STArray total transcriptional chip of Affymetrix company, using the random variance model (RVM), using P? 0.05 and FDR? 0.05 as the criterion, differential gene screening was carried out. The genes related to iron metabolism were found by GO taxonomy, and the gene expression trend was consistent with the change trend of liver iron load.
3, the expression of hepcidin, TfR1 and sfxn1mRNA in rat liver
The real time fluorescence quantitative PCR method was used to determine the changes in the expression of hepcidin, transferrin receptor 1 (transferrin receptor1, TfR1), and sideroflexin1 (sfxn1) in rat liver.
4, the contents of hepcidin, TfR1 and sfxn1 proteins in rat liver tissues were measured.
The contents of hepcidin, TfR1 and sfxn1 in liver of each group were measured by immuno imprinting.
Three, the effect of SFXN1 on the major regulatory molecules of iron metabolism in liver cells.
1, cell culture
HepG2 hepatocyte strain, DMEM culture medium containing 10% fetal bovine serum, was cultured at 37 C and 5%CO2.
2, Sfxn1siRNA transfected cells
Sfxn1siRNA was transfected into HepG2 cells according to instructions, and 48h. was cultured in incubator.
3, determination of hepatocyte sfxn1, TfR1, hepcidin, IRP1, IRP2mRNA
The expression levels of sfxn1, TfR1, hepcidin, IRP1 and IRP2mRNA in HepG2 cells were measured by real-time fluorescence quantitative PCR.
4, determination of protein sfxn1, TfR1, hepcidin, IRP1 and IRP2 in hepatocytes
The contents of sfxn1, TfR1, hepcidin, IRP1 and IRP2 in HepG2 cells were determined by Western blot.
Four, statistics and processing of data
SPSS16.0 statistical software package was used to analyze the experimental data. Two independent sample t- tests were used between the two groups; the multiple groups were compared by single factor analysis of variance, and 22 of the samples were compared with the LSD-t test, and the Dunnett method was compared with the control group. Dunnett 's was used when the variance was uneven. C test. The experimental data were expressed by mean + standard deviation (x + s), and P0.05 thought it was statistically significant.
Result
Changes in serum iron and liver iron contents in rats with psychological stress
1, the changes of serum CORT, ACTH and NE levels in rats with psychological stress.
The contents of CORT, ACTH and NE in psychological stress rats were significantly higher than those in the control group, indicating that the psychological stress model was successfully produced.
2, changes of iron content in the liver of rats with psychological stress
With the prolongation of stress time, the iron content of liver tissue in rats increased gradually. The iron content in liver of the rats in the psychological stress group increased by 53.02% (P0.05) and 3 days in the psychological stress group. The iron content of the rats in the psychological stress group had no significant difference between the control group and the control group on the 7 day (P0.05).
3, change of iron content in serum of psychological stress rats
There was no significant difference in serum iron content between the 1 days and 3 days of psychological stress group with the control group (P0.05). Compared with the control group, the serum iron content of the rats in the psychological stress group decreased by 29.21% in 7 days, and the difference was statistically significant (P0.05).
4, the change of serum transferrin saturation in rats with psychological stress.
Compared with the control group, the serum transferrin saturation of the rats in the 7 day psychological stress group decreased by 32.2% (P0.05), and the serum transferrin saturation of the rats in the psychological stress group was 7.87% and 19.85%, respectively, on 1 and 3 days, but there was no statistical significance (P0.05).
Two, the changes of SFXN1 and iron metabolism related molecules in the liver of rats with psychological stress.
1, gene chip screening of genes related to iron metabolism in rats with psychological stress.
In the process of psychological stress, there are 2246 significant differences in the transcriptional level of liver gene mRNA in rats. There are seven differentially differentially expressed genes related to iron metabolism for these differentially expressed genes, which are Hamp, Tfrc, sfxn1, GDF2, IREB2, NUBP1, and NUBP2.sfxn1 gene expression trend analysis show that the trend of the change and liver iron precipitation The trend of product change is consistent.
2, the expression of hepcidin and TfR1mRNA in the liver of rats with psychological stress.
After 7 days of psychological stress, the expression level of hepcidin mRNA in the rat liver was significantly higher than that in the control group, 1 days and 3 days in the psychological stress group (P0.01), and the expression level of TfR1mRNA in seventh days of psychological stress was higher than that of the control, 1 days, and 3 days of psychological stress group, and had statistical significance.
3, the expression of hepcidin and TfR1 protein in the liver of rats with psychological stress.
The expression level of hepcidin protein in the 7 day psychological stress group was significantly higher than that of the control group, 1 days, and the 3 days of psychological stress group increased significantly (P0.01).TfR1 protein increased gradually after stress. The expression level of TfR1 protein in the psychological stress group was statistically significant (P0.01) on the 7 day stress group.
4, changes in the expression of sfxn1 in the liver of rats with psychological stress
After stress, the level of sfxn1mRNA increased gradually. The 3 day stress group was significantly higher than the control group and the control group and the 1 day stress group (P0.01). The expression level of sfxn1mRNA in the 7 day stress group was higher than the control group, 1 days and 3 days of psychological stress group, and had statistical significance (P0.01).
Sfxn1 protein increased gradually after stress. On the 7 day, the expression level of psychological stress group was 66% higher than that of the control group (P0.01).
Three, the effect of SFXN1 on the major regulatory molecules of iron metabolism in liver cells.
1, the effect of Sfxn1siRNA transfection on the expression of sfxn1mRNA in HepG2 cells.
The expression of sfxn1mRNA in HepG2 cells transfected with sfxn1siRNA was lower than that in untransfected control group (P0.05).
2, the effect of Sfxn1siRNA transfection on the expression of TfR1 and hepcidin mRNA in HepG2 cells.
After Sfxn1siRNA transfection of HepG2 cells for 48 hours, the expression of TfR1mRNA in the transfected group decreased by 78% (P0.01).Hepcidin mRNA than that in the control group, and there was no difference compared with the control group.
3, the effect of Sfxn1siRNA transfection on the expression of IRP1 and IRP2mRNA in HepG2 cells.
After Sfxn1siRNA transfection of HepG2 cells for 48 hours, the expression of IRP1mRNA in the transfected group was 80% lower than that in the untransfected group (P0.05). There was no difference in the expression of IRP2mRNA in the transfected group compared with the control group.
4, Sfxn1siRNA transfection reduced the expression of sfxn1 protein in HepG2 cells.
After transfection of HepG2 cells with Sfxn1siRNA, the expression of sfxn1 protein decreased (P0.01).
5, the effect of Sfxn1siRNA interference on the expression of TfR1 and hepcidin protein in HepG2 cells.
Sfxn1siRNA transfected HepG2 cells reduced the expression of TfR1 protein (P0.01) and the protein expression of.Hepcidin was not affected.
6, the effect of Sfxn1siRNA interference on the expression of IRP1 and IRP2 protein in HepG2 cells.
After transfection of HepG2 cells with Sfxn1siRNA, the expression of IRP1 protein decreased (P0.01) and.IRP2 expression did not change.
conclusion
1. psychological stress for 7 consecutive days can cause liver iron accumulation in rats.
2. gene chip found that Hamp, Tfrc, sfxn1, GDF2, IREB2, NUBP1, NUBP2 in psychological stress changed.Hepcidin, TfR1, sfxn1 mRNA and protein levels increased with the increase of the number of stress days.
3. sfxn1 can affect the expression of TfR1
In summary, psychological stress may increase the expression of transferrin receptor 1 through sfxn1, and increase iron intake in liver cells, resulting in liver iron accumulation.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R151.2
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