【摘要】：鐵是機(jī)體含量最豐富的金屬元素之一，含鐵蛋白在體內(nèi)發(fā)揮重要的生理功能，參與氧的轉(zhuǎn)運(yùn)，細(xì)胞呼吸、物質(zhì)代謝、轉(zhuǎn)錄調(diào)節(jié)和DNA修復(fù)。機(jī)體鐵缺乏會產(chǎn)生缺鐵性貧血和多種代謝障礙，然而過量的鐵在體內(nèi)能通過Fenton反應(yīng)引起脂質(zhì)過氧化，進(jìn)一步損傷蛋白質(zhì)和（或）核酸。機(jī)體鐵負(fù)荷與癌癥、冠心病、帕金森病、糖尿病等多種疾病的發(fā)生有關(guān)。肝臟是機(jī)體儲存鐵的主要器官，，也是鐵過量損傷的主要器官，有大量研究資料證實(shí)，肝鐵過負(fù)荷會導(dǎo)致肝細(xì)胞壞死，纖維化和肝腫瘤，還能引起胰島素抵抗、增加膽固醇的合成和沉積。因此探討鐵過負(fù)荷的原因，尋找防治的潛在靶點(diǎn)成為醫(yī)學(xué)研究的熱點(diǎn)。目前已證實(shí)HFE、HJV、TfR2等基因突變導(dǎo)致遺傳性血色素沉著病，反復(fù)輸血、膳食鐵攝入過多等因素都可以引起機(jī)體鐵過負(fù)荷，但是對于大多數(shù)疾病存在鐵過負(fù)荷的原因尚未完全闡明。 心理應(yīng)激是指機(jī)體對外界有害物、威脅、挑戰(zhàn)經(jīng)認(rèn)識評價(jià)后，知其將危害個人的生存和地位時，所產(chǎn)生的生理、心理和行為反應(yīng)�，F(xiàn)代生活中，人們在工作和生活中承受的負(fù)荷越來越大，精神緊張和心理壓力已成為重要的應(yīng)激源，心理因素對人體健康的影響正引起研究者廣泛的關(guān)注。課題組前期的研究發(fā)現(xiàn)，心理應(yīng)激可引起實(shí)驗(yàn)動物肝鐵蓄積。其中部分原因可能是通過IL-6上調(diào)hepcidin及糖皮質(zhì)激素對IRP1的調(diào)節(jié)，由于心理應(yīng)激動物肝鐵蓄積十分明顯，是否還有其它的因素導(dǎo)致肝鐵蓄積有待于進(jìn)一步研究。 為了深入分析心理應(yīng)激導(dǎo)致肝鐵蓄積的可能原因，本研究采用基因芯片技術(shù)篩選心理應(yīng)激大鼠肝臟的差異表達(dá)基因，并根據(jù)基因表達(dá)趨勢分析與肝鐵負(fù)荷變化趨勢相一致的基因，尋找與肝鐵代謝變化有關(guān)的分子。結(jié)果發(fā)現(xiàn)在心理應(yīng)激情況下，hepcidin、TfR1等已知的與鐵代謝有關(guān)的分子發(fā)生了變化，還發(fā)現(xiàn)SFXN1的表達(dá)增強(qiáng)。SFXN1也稱為三羧基攜帶蛋白TCC，是sideroflexin家族成員之一，定位于線粒體膜上，是真核生物的一種進(jìn)化保守蛋白。有研究資料表明，SFXN1可能具有轉(zhuǎn)運(yùn)有利于鐵利用的某種成分進(jìn)入線粒體的功能，flexed-tail小鼠SFXN1發(fā)生了移碼突變，表現(xiàn)出紅細(xì)胞線粒體內(nèi)鐵沉積的特征。線粒體是細(xì)胞電子傳遞和能量代謝最為旺盛的細(xì)胞器，線粒體內(nèi)鐵代謝紊亂會嚴(yán)重地影響到整個細(xì)胞的鐵代謝。因此，SFXN1是否對細(xì)胞鐵代謝相關(guān)分子產(chǎn)生一定的影響，從而參與心理應(yīng)激反應(yīng)肝鐵負(fù)荷的發(fā)生，我們進(jìn)行了進(jìn)一步實(shí)驗(yàn)研究。 研究目的 利用基因芯片篩選心理應(yīng)激情況下大鼠肝臟的差異表達(dá)基因，并在此基礎(chǔ)上尋找可能參與心理應(yīng)激鐵代謝異常的基因，為闡明心理應(yīng)激導(dǎo)致肝鐵負(fù)荷提供新的研究思路。 研究方法 一、心理應(yīng)激大鼠血清鐵和肝鐵含量變化 1、實(shí)驗(yàn)動物分組 健康雄性SD大鼠48只（體重120±10g），購自中英合資上海西普爾-必凱實(shí)驗(yàn)動物有限公司。按體重隨機(jī)分為對照組、1天、3天、7天心理應(yīng)激組4組，每組8只，其余作為電擊組。自由飲食，動物實(shí)驗(yàn)室條件為：溫度24℃士1℃，濕度50％～60％，光照自然晝夜節(jié)律變化。 2、制作心理應(yīng)激模型 大鼠心理應(yīng)激模型制作采用Communication box system。該裝置由透明丙烯酸板制成，所有小室的一半鋪絕緣板（A室），另一半不鋪絕緣板（B室）。B室的大鼠每天足底電擊30分鐘，電壓為80V，大鼠被電擊后尖叫、跳躍、排便；相鄰A室的大鼠通過聽覺、視覺和嗅覺產(chǎn)生心理應(yīng)激。 3、血清CORT、ACTH及NE測定 按檢測試劑盒說明測定血清CORT、ACTH及NE的濃度。 4、測定肝鐵、血清鐵含量 濕法消化肝組織，原子吸收分光光度計(jì)火焰法測定肝鐵和血清鐵含量。 5、血清轉(zhuǎn)鐵蛋白飽和度的測定 采用血清鐵和血清總鐵結(jié)合力試劑盒測定。 二、心理應(yīng)激大鼠肝臟SFXN1及鐵代謝相關(guān)分子的變化 1、實(shí)驗(yàn)動物分組及處理 第一部分的實(shí)驗(yàn)動物，每組隨機(jī)抽取三只大鼠，選擇肝組織進(jìn)行基因芯片分析。 2、基因芯片分析 采用Affymetrix公司的Rat Exon1.0STArray全轉(zhuǎn)錄組表達(dá)譜芯片，利用隨機(jī)方差模型（RVM），以P㩳0.05和FDR㩳0.05為判斷標(biāo)準(zhǔn)，進(jìn)行差異基因篩選。用GO分類法查找與鐵代謝有關(guān)的基因，并根據(jù)基因表達(dá)趨勢分析與肝鐵負(fù)荷變化趨勢相一致的基因。 3、大鼠肝臟hepcidin、TfR1、sfxn1mRNA表達(dá)的變化 采用實(shí)時熒光定量PCR法測定大鼠肝臟內(nèi)鐵調(diào)素(hepcidin)、轉(zhuǎn)鐵蛋白受體1（transferrin receptor1，TfR1）、sideroflexin1（sfxn1）的mRNA表達(dá)變化。 4、測定大鼠肝組織hepcidin、TfR1、sfxn1蛋白含量 免疫印記法測定各組大鼠肝臟hepcidin、TfR1、sfxn1的蛋白含量。 三、 SFXN1對肝細(xì)胞鐵代謝主要調(diào)節(jié)分子的影響 1、細(xì)胞培養(yǎng) HepG2肝細(xì)胞株，含10%胎牛血清的DMEM培養(yǎng)液37℃、5%CO2培養(yǎng)。 2、Sfxn1siRNA轉(zhuǎn)染細(xì)胞 按說明書將sfxn1siRNA轉(zhuǎn)染HepG2細(xì)胞后，培養(yǎng)箱中培養(yǎng)48h。 3、肝細(xì)胞sfxn1、TfR1、hepcidin、IRP1、IRP2mRNA測定 采用實(shí)時熒光定量PCR法測定HepG2細(xì)胞sfxn1、TfR1、hepcidin、IRP1、IRP2mRNA的表達(dá)水平。 4、肝細(xì)胞sfxn1、TfR1、hepcidin、IRP1、IRP2的蛋白測定 采用Western blot法測定HepG2細(xì)胞sfxn1、TfR1、hepcidin、IRP1、IRP2的蛋白含量。 四、數(shù)據(jù)的統(tǒng)計(jì)與處理 采用SPSS16.0統(tǒng)計(jì)軟件包對實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。兩組間比較采用兩獨(dú)立樣本t-檢驗(yàn)；多組間比較采用單因素方差分析，方差齊時各組樣本均數(shù)間兩兩比較采用LSD-t檢驗(yàn)，各實(shí)驗(yàn)組間與對照組比較采用Dunnett法。方差不齊時采用Dunnett’s C檢驗(yàn)。實(shí)驗(yàn)數(shù)據(jù)以平均數(shù)±標(biāo)準(zhǔn)差（x±s）表示，p0.05認(rèn)為有統(tǒng)計(jì)學(xué)意義。 結(jié)果 一、心理應(yīng)激大鼠血清鐵和肝鐵含量變化 1、心理應(yīng)激大鼠血清CORT、ACTH與NE含量變化 心理應(yīng)激大鼠血清CORT、ACTH、NE含量較對照組明顯升高，說明心理應(yīng)激模型成功制作。 2、心理應(yīng)激大鼠肝臟鐵含量變化 隨著應(yīng)激時間的延長，大鼠肝組織鐵含量逐漸升高，7天心理應(yīng)激組大鼠肝臟鐵含量與對照組比較上升了53.02%（P0.05）。1天和3天心理應(yīng)激組大鼠肝鐵含量與對照組比較無統(tǒng)計(jì)學(xué)意義（P0.05）。 3、心理應(yīng)激大鼠血清鐵含量變化 1天和3天心理應(yīng)激組大鼠血清鐵含量與對照組比較無統(tǒng)計(jì)學(xué)意義（P0.05）,與對照組比較，7天心理應(yīng)激組大鼠血清鐵含量下降了29.21%，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 4、心理應(yīng)激大鼠血清轉(zhuǎn)鐵蛋白飽和度的變化 與對照組比較，7天心理應(yīng)激組大鼠血清轉(zhuǎn)鐵蛋白飽和度下降了32.2%（P0.05）；1天和3天心理應(yīng)激組大鼠血清轉(zhuǎn)鐵蛋白飽和度較對照組分別下降了7.87%、19.85%，但無統(tǒng)計(jì)學(xué)意義（P0.05）。 二、心理應(yīng)激大鼠肝臟SFXN1及鐵代謝相關(guān)分子的變化 1、基因芯片篩選心理應(yīng)激大鼠肝臟鐵代謝相關(guān)差異基因 在心理應(yīng)激過程中，大鼠肝臟基因mRNA轉(zhuǎn)錄水平有明顯差異的為2246個。對這些差異表達(dá)基因進(jìn)行生物信息學(xué)分析，與鐵代謝有關(guān)的差異基因有七個，分別為Hamp、Tfrc、sfxn1、GDF2、IREB2、NUBP1、NUBP2。sfxn1基因的表達(dá)趨勢分析顯示其變化趨勢與肝鐵沉積變化趨勢一致。 2、心理應(yīng)激大鼠肝臟hepcidin、TfR1mRNA的表達(dá)變化 7天心理應(yīng)激后大鼠肝臟hepcidin mRNA的表達(dá)水平較對照、1天、3天心理應(yīng)激組均有顯著升高（P0.01）；心理應(yīng)激第七天TfR1mRNA的表達(dá)水平比對照、1天、3天心理應(yīng)激組升高，并有統(tǒng)計(jì)學(xué)意義。 3、心理應(yīng)激大鼠肝臟hepcidin、TfR1的蛋白表達(dá)變化 7天心理應(yīng)激組hepcidin蛋白的表達(dá)水平較對照、1天、3天心理應(yīng)激組均有顯著升高（P0.01）。TfR1蛋白在應(yīng)激后逐漸升高，7天心理應(yīng)激組TfR1蛋白的表達(dá)水平與對照組比較有統(tǒng)計(jì)學(xué)意義（P0.01）。 4、心理應(yīng)激大鼠肝臟sfxn1的表達(dá)變化 應(yīng)激后sfxn1mRNA的水平逐漸升高，3天應(yīng)激組與對照組、1天應(yīng)激組比較有統(tǒng)計(jì)學(xué)意義（P0.01）；7天應(yīng)激組sfxn1mRNA的表達(dá)水平比對照、1天、3天心理應(yīng)激組升高，并有統(tǒng)計(jì)學(xué)意義（P0.01）。 Sfxn1蛋白在應(yīng)激后逐漸升高，7天心理應(yīng)激組的表達(dá)水平較對照組升高66%（P0.01）。 三、SFXN1對肝細(xì)胞鐵代謝主要調(diào)節(jié)分子的影響 1、Sfxn1siRNA轉(zhuǎn)染對HepG2細(xì)胞sfxn1mRNA表達(dá)的影響 HepG2細(xì)胞轉(zhuǎn)染sfxn1siRNA后sfxn1mRNA的表達(dá)量較未轉(zhuǎn)染的對照組降低（P0.05）。 2、Sfxn1siRNA轉(zhuǎn)染對HepG2細(xì)胞TfR1、 hepcidin mRNA表達(dá)的影響 Sfxn1siRNA轉(zhuǎn)染HepG2細(xì)胞48小時后，轉(zhuǎn)染組TfR1mRNA的表達(dá)量較未轉(zhuǎn)染組降低了78%（P0.01）。Hepcidin mRNA的表達(dá)量與對照組比較無區(qū)別。 3、Sfxn1siRNA轉(zhuǎn)染對HepG2細(xì)胞IRP1、 IRP2mRNA表達(dá)的影響 Sfxn1siRNA轉(zhuǎn)染HepG2細(xì)胞48小時后，轉(zhuǎn)染組IRP1mRNA的表達(dá)量較未轉(zhuǎn)染組降低了80%（P0.05）。轉(zhuǎn)染組IRP2mRNA的表達(dá)量與對照組比較無差別。 4、Sfxn1siRNA轉(zhuǎn)染降低HepG2細(xì)胞sfxn1蛋白表達(dá) Sfxn1siRNA轉(zhuǎn)染HepG2細(xì)胞后使sfxn1蛋白表達(dá)下降（P0.01）。 5、Sfxn1siRNA干擾對HepG2細(xì)胞TfR1、 hepcidin蛋白表達(dá)的影響 Sfxn1siRNA轉(zhuǎn)染HepG2細(xì)胞后使TfR1蛋白表達(dá)下降（P0.01）。hepcidin的蛋白表達(dá)不受影響。 6、Sfxn1siRNA干擾對HepG2細(xì)胞IRP1、 IRP2蛋白表達(dá)的影響 Sfxn1siRNA轉(zhuǎn)染HepG2細(xì)胞后使IRP1蛋白表達(dá)下降（P0.01）。IRP2的表達(dá)未發(fā)生改變。 結(jié)論 1.連續(xù)7天心理應(yīng)激可以引起大鼠肝鐵蓄積。 2.基因芯片發(fā)現(xiàn)心理應(yīng)激過程中Hamp、Tfrc、sfxn1、GDF2、IREB2、NUBP1、NUBP2共7個與鐵代謝相關(guān)的基因發(fā)生了改變。hepcidin、TfR1、sfxn1的mRNA和蛋白水平隨著應(yīng)激天數(shù)的增加不斷上升。 3. sfxn1能影響TfR1的表達(dá) 綜上所述，心理應(yīng)激時可能通過sfxn1引起轉(zhuǎn)鐵蛋白受體1表達(dá)增加，肝細(xì)胞攝入鐵增加，導(dǎo)致肝鐵蓄積。
[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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R151.2

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