本文選題：心理應(yīng)激 + 血清鐵��； 參考：《第二軍醫(yī)大學(xué)》2007年碩士論文

【摘要】： 鐵是合成血紅蛋白的重要原料。大量研究資料表明血清鐵含量下降可影響血紅蛋白的合成,進(jìn)而影響紅細(xì)胞的形成與成熟,甚至引起缺鐵性貧血[1,2]。據(jù)WHO報(bào)道,全世界患缺鐵性貧血人數(shù)約有10億。我國(guó)缺鐵性貧血的發(fā)病率也很高,孫建琴等報(bào)道上海兒童、青少年人群高達(dá)19.9%[3]。由于缺鐵性貧血可影響人體多種生理功能和生殖能力[4,5],因此該病的防治一直受到人們極大的關(guān)注。引發(fā)缺鐵性貧血的主要原因是鐵攝入不足和鐵丟失過(guò)多[7]。但也有研究發(fā)現(xiàn),部分人群即使在膳食鐵供給充足且無(wú)過(guò)量丟失的情況下,缺鐵性貧血的發(fā)病率依然居高不下[8];也就是說(shuō)缺鐵性貧血的高發(fā)可能存在膳食鐵攝入量以外的影響因素。但迄今為止,研究者對(duì)造成高膳食鐵攝入量和高缺鐵性貧血發(fā)生率這一矛盾現(xiàn)象的原因還不清楚[9-11]。 我們?cè)谝酝芯恐邪l(fā)現(xiàn),應(yīng)激可以導(dǎo)致血清鐵大幅度下降,例如:加速度刺激可導(dǎo)致人體血清鐵下降52.3%[12];熱暴露可引起大鼠血清鐵下降31%[13]。國(guó)外也有不少同類研究報(bào)道,例如:Navas FJ等報(bào)道大運(yùn)動(dòng)量訓(xùn)練可引起運(yùn)動(dòng)員血清鐵下降[14];Smith SM等發(fā)現(xiàn)航天飛行可導(dǎo)致宇航員血清鐵下降[15];Nikolova- Todorova Z等提出手術(shù)創(chuàng)傷也可造成病人血清鐵下降[16]。但迄今為止,尚無(wú)關(guān)于心理應(yīng)激是否會(huì)導(dǎo)致血清鐵下降,以及心理應(yīng)激引起的血清鐵下降是否會(huì)影響紅細(xì)胞生成的報(bào)道。本研究對(duì)不同心理應(yīng)激周期SD大鼠血清鐵、鐵調(diào)節(jié)蛋白及紅細(xì)胞生成的變化進(jìn)行了觀察。 目的 了解心理應(yīng)激對(duì)大鼠造血系統(tǒng)鐵代謝及紅細(xì)胞生成的影響,為闡明心理應(yīng)激與紅細(xì)胞代謝關(guān)系提供實(shí)驗(yàn)基礎(chǔ)。 方法 1.實(shí)驗(yàn)動(dòng)物分組 雄性SD大鼠,體質(zhì)量80±5g,上海西普爾-必凱公司提供。單籠飼養(yǎng),自由飲食,室溫24±1℃,相對(duì)濕度50～60%,12h/12h光暗周期節(jié)律。適應(yīng)3d后按體重隨機(jī)分成空白對(duì)照組(control group, CG)、心理應(yīng)激組(psychological stress group, PSG)和足底電擊組(foot-shock stress group, FSG),每組再分為3d、7d和14d三個(gè)亞組。 2.建立SD大鼠心理應(yīng)激模型 按照文獻(xiàn)[17]采用Communication Box System制作大鼠心理應(yīng)激模型。Communication Box System由透明丙烯酸板組成,一半小室(A)底部鋪板絕緣,另一半小室(B室)通電。在B室的實(shí)驗(yàn)大鼠接受30min/d,足底電擊(電壓90V,電流0.80mA);電擊組大鼠接受電擊時(shí)跳躍,尖叫,在A室的實(shí)驗(yàn)大鼠通過(guò)視覺聽覺產(chǎn)生恐懼的心理反應(yīng),即為心理應(yīng)激大鼠模型。按計(jì)劃完成不同周期應(yīng)激暴露后,各組大鼠立刻斷頭處死取組織標(biāo)本備用。 3.大鼠血清、肝臟和脾臟鐵含量的測(cè)定 采用原子吸收分光光度計(jì)(日本日立公司Z-2000型)火焰法測(cè)定血清鐵(serum iron, SI)和肝脾鐵含量。100 u g/mL鐵標(biāo)準(zhǔn)貯備液(GBW08616)由國(guó)家標(biāo)準(zhǔn)物質(zhì)中心提供。 4.大鼠紅細(xì)胞參數(shù)的測(cè)定 使用KX-21血細(xì)胞分析儀(SYSMEX,日本)測(cè)定血紅蛋白(hemoglobin,Hb)濃度、紅細(xì)胞計(jì)數(shù)(red blood cell count ,RBC)、紅細(xì)胞壓積(hematocrit, Hct)、平均紅細(xì)胞體積(mean corpuscular volume,MCV)和紅細(xì)胞分布寬度(red blood cell distribution width,RDW)。 5.大鼠骨髓鐵含量的測(cè)定 取大腿股骨頭新鮮骨髓制成涂片,于24h內(nèi)用酸性亞鐵氰化鉀進(jìn)行染色,顯微鏡下觀察鐵染色顆粒,進(jìn)行半定量和定性分析。 6.大鼠血清鐵調(diào)節(jié)蛋白及促紅細(xì)胞生成素含量的測(cè)定 應(yīng)用ELISA方法測(cè)定大鼠血清鐵蛋白(serum ferritin, SF)、血清轉(zhuǎn)鐵蛋白受體(serum transferring receptor, sTfR)和血清促紅細(xì)胞生成素(serum erythropoietin, sEPO)。ELISA試劑盒(購(gòu)自RD Sysytems Inc,USA)組成:96孔酶標(biāo)板、底物A、底物B、濃縮洗滌液、終止液、酶聯(lián)物、標(biāo)準(zhǔn)品一套。測(cè)試儀器:自動(dòng)酶標(biāo)儀(SUNRISE,美國(guó))。 7.統(tǒng)計(jì)方法 實(shí)驗(yàn)數(shù)據(jù)采用SPSS10.0統(tǒng)計(jì)軟件包采用one-way ANOVA檢驗(yàn)進(jìn)行數(shù)據(jù)分析,實(shí)驗(yàn)數(shù)據(jù)以平均數(shù)±標(biāo)準(zhǔn)差( X±S)表示,顯著性水平為P0.05,非常顯著性水平為P0.01。 結(jié)果 1.心理應(yīng)激對(duì)大鼠血清鐵含量的影響 與對(duì)照組相比,3d、7d和14d心理應(yīng)激組大鼠的SI分別下降28.4%、27.7%和34.0%(P0.05),3d、7d和14d足底電擊組大鼠的SI則分別下降28.5%、18.3%和40.7%,(P0.05)。相同應(yīng)激周期的心理應(yīng)激組與足底電擊組大鼠相比,SI含量無(wú)顯著差異(P0.05)。 2.心理應(yīng)激對(duì)大鼠骨髓鐵含量的影響 酸性亞鐵氰化鉀染色顯示,與對(duì)照組比較,心理應(yīng)激7d組大鼠和足底電擊7d組大鼠骨髓細(xì)胞外鐵含量明顯減少,心理應(yīng)激14d組大鼠和足底電擊14d組大鼠骨髓細(xì)胞外鐵含量進(jìn)一步下降,甚至缺如;而相同應(yīng)激周期的心理應(yīng)激組與足底電擊組相比,無(wú)顯著差異。 3.心理應(yīng)激對(duì)大鼠血清鐵調(diào)節(jié)蛋白的影響 與對(duì)照組相比,心理應(yīng)激3d大鼠的SF顯著升高(P0.05);而連續(xù)心理應(yīng)激7d和14d組大鼠的SF分別下降35.9%和28.3%(P0.05),同時(shí)伴有sTfR分別升高42.2%和31.5%(P0.01)。而相同應(yīng)激周期的足底電擊組大鼠血清鐵調(diào)節(jié)蛋白的變化和心理應(yīng)激組基本一致(P0.05)。 4.心理應(yīng)激對(duì)大鼠紅細(xì)胞參數(shù)的影響 與對(duì)照組相比,7d和14d心理應(yīng)激組大鼠的Hb分別下降10.0%和12.8%(P0.05)、RBC分別下降4.7%和9.8%(P0.05)、Hct分別下降7.0%和12.9%(P0.05)、MCV分別下降1.6%和7.3%(P0.05),而RDW則分別增加11.2%和22.5%(P0.05);7d和14d足底電擊組大鼠的Hb分別下降8.7%和9.9%(P0.05)、RBC分別下降6.1%和10.5%(P0.05)、Hct分別下降5.4%和11.4%(P0.05)、MCV分別下降3.6%和1.5%(P0.05),而RDW則分別增加3.6%和6.1%(P0.05)。相同應(yīng)激周期的心理應(yīng)激組大鼠紅細(xì)胞各參數(shù)和足底電擊組大鼠相比,均無(wú)顯著差異(P0.05)。 5.心理應(yīng)激對(duì)大鼠進(jìn)食量和體重的影響 與對(duì)照組相比,心理應(yīng)激3d、7d或14d的大鼠進(jìn)食量和體重均無(wú)明顯變化(P0.05)。相同應(yīng)激周期的心理應(yīng)激組大鼠進(jìn)食量和體重與足底電擊組變化基本一致(P0.05)。 6.心理應(yīng)激對(duì)大鼠肝鐵和脾鐵含量的影響 與對(duì)照組相比,心理應(yīng)激7d和14d組大鼠肝鐵含量分別升高41.3%和24.9% (P0.05),脾鐵含量分別升高12.3%和10.4%(P0.05);足底電擊7d和14d組大鼠肝鐵含量分別升高24.7%和10.3% (P0.05),脾鐵含量分別升高15.1%和17.8%(P0.05)。相同應(yīng)激周期的心理應(yīng)激組大鼠肝、脾鐵含量和足底電擊組大鼠相比,無(wú)顯著差異(P0.05)。 7.心理應(yīng)激對(duì)大鼠促紅細(xì)胞生成素的影響 與對(duì)照組相比,14d時(shí)心理應(yīng)激組和足底電擊組sEPO分別下降12.3%和35.8%(P0.05); 14d足底電擊組與心理應(yīng)激組相比,sEPO顯著升高26.6%(P0.05)。 結(jié)論 1.連續(xù)心理應(yīng)激3d可導(dǎo)致大鼠的血清鐵含量明顯下降,而連續(xù)心理應(yīng)激14d可導(dǎo)致大鼠血清鐵含量進(jìn)一步下降。心理應(yīng)激大鼠血清鐵含量的變化趨勢(shì)與軀體應(yīng)激(足底電擊)大鼠基本一樣。 2.心理應(yīng)激可以導(dǎo)致大鼠骨髓鐵含量顯著降低。連續(xù)應(yīng)激14d時(shí),SD大鼠骨髓細(xì)胞外鐵染色減少程度較應(yīng)激7d時(shí)更顯著。上述結(jié)果表明,心理應(yīng)激大鼠血清鐵水平降低可進(jìn)而影響其骨髓鐵含量。 3.較長(zhǎng)時(shí)間的心理應(yīng)激(連續(xù)應(yīng)激7d)可導(dǎo)致大鼠血清鐵蛋白含量顯著下降,同時(shí)伴有血清轉(zhuǎn)鐵蛋白受體含量顯著升高。綜合心理應(yīng)激可影響血清鐵和骨髓鐵含量等觀察結(jié)果,提示心理應(yīng)激引起的血清鐵含量降低,可導(dǎo)致機(jī)體貯存鐵和骨髓鐵減少,進(jìn)而影響到骨髓的紅細(xì)胞生成。至于應(yīng)激3d組大鼠SF含量增加,可能與SF為急性時(shí)相反應(yīng)蛋白有關(guān),即SF在應(yīng)激早期升高;但隨著應(yīng)激狀態(tài)的延續(xù),血清鐵持續(xù)降低,SF含量逐漸下降。確切原因有待進(jìn)一步探討。 4.心理應(yīng)激可導(dǎo)致SD大鼠的RBC、Hb、HCt等參數(shù)明顯下降,下降幅度隨著應(yīng)激周期的延長(zhǎng)而增加;同時(shí)伴有MCV顯著下降,RDW顯著升高,RDW/MCV顯著升高(RDW/MCV比值升高,在臨床中作為小細(xì)胞低色素性貧血的診斷指標(biāo)之一)。綜合本課題研究結(jié)果表明,長(zhǎng)時(shí)間處于心理應(yīng)激狀態(tài),可因?yàn)檠彖F和骨髓鐵的降低而在一定程度上影響紅細(xì)胞的生成。 5.本實(shí)驗(yàn)結(jié)果顯示,心理應(yīng)激對(duì)大鼠進(jìn)食量和體重均無(wú)明顯影響,提示心理應(yīng)激大鼠血清鐵降低可能與飲食鐵攝入量無(wú)關(guān)。然而,心理應(yīng)激是否會(huì)影響飲食鐵吸收率則需要進(jìn)一步研究。 6.一般情況下,紅細(xì)胞生成絕對(duì)不足或相對(duì)不足可導(dǎo)致sEPO含量升高,本實(shí)驗(yàn)中心理應(yīng)激組大鼠在連續(xù)應(yīng)激14d時(shí)sEPO卻顯著降低。心理應(yīng)激導(dǎo)致sEPO下降的原因,以及心理應(yīng)激導(dǎo)致的sEPO降低是否為影響紅細(xì)胞生成的原因之一,均有待于進(jìn)一步探討。 7.連續(xù)應(yīng)激可造成大鼠肝鐵和脾鐵含量顯著升高。提示心理應(yīng)激條件下,大鼠體內(nèi)鐵的重新分布可能是其血清鐵含量降低的原因之一。
[Abstract]:Iron is an important raw material for the synthesis of hemoglobin . A large amount of research shows that the decrease of serum iron content can affect the synthesis of hemoglobin , which can affect the formation and maturation of red blood cells and even lead to iron deficiency anemia . According to WHO , the incidence of iron deficiency anemia in the world is also high , Sun Jian - qin et al . reported Shanghai children , and the youth population was as high as 19 . 9 % . Since iron deficiency anemia can affect various physiological functions and reproductive capacity of human body , the prevention and treatment of iron deficiency has been a great concern . The main reason for iron deficiency anemia is that iron intake is insufficient and iron loss is too much . However , it has been found that the incidence of iron deficiency anemia remains high even in the absence of adequate supply of dietary iron and no excess loss ; that is , the high incidence of iron deficiency anemia may be a factor other than dietary iron intake . However , to date , the researchers are not aware of the causes of this paradox of high dietary iron intake and high iron deficiency anemia .






In our previous studies , stress could lead to a significant decrease in serum iron , for example : acceleration stimulation could lead to a decrease of serum iron in the human body by 52.3 % ; heat exposure could lead to a decrease in serum iron in rats by 31 % . There are many similar studies abroad , such as : Navas FJ et al . reports that large amounts of exercise training can lead to a decrease in iron in the serum iron of athletes ; Smith SM , et al . found that space flight could lead to a reduction in serum iron in the astronauts , which could lead to decreased serum iron reduction of the patient . But to date , there is no report on whether psychological stress can lead to the decrease of serum iron , and whether the decrease of serum iron caused by psychological stress can affect the formation of red blood cells . This study has made an observation on the changes of serum iron , iron - regulated protein and erythrocyte formation in SD rats with different psychological stress periods .






Purpose






To understand the effects of psychological stress on iron metabolism and erythrocyte formation in hematopoietic system in rats , and to provide an experimental basis for clarifying the relationship between psychological stress and erythrocyte metabolism .






method






1 . Group of experimental animals






Male SD rats were randomly divided into three groups : control group ( CG ) , psychological stress group ( PSG ) and foot - shock stress group .






2 . Establishment of SD rat psychological stress model






The psychological stress model of rats was made by using Communication Box System . Communication Box System was made up of transparent acrylic plate , half cell ( A ) was plated with insulation , and the other half cell ( B chamber ) was energized . The experimental rats in B room were subjected to shock ( voltage 90V , current 0.80 mA ) .






3 . Determination of iron content in serum , liver and spleen of rats






6 . In general , the absolute deficiency or relative deficiency of red blood cells could result in the increase of sEPO content . In this experiment , sEPO decreased significantly at 14 days after continuous stress . The reason for the decrease of sEPO caused by psychological stress , and whether the decrease of sEPO caused by psychological stress was one of the reasons affecting the formation of erythrocytes .






4 . Determination of red blood cell parameters in rats






Hemoglobin ( hemoglobin , Hb ) concentration , red blood cell count ( RBC ) , red blood cell count ( hematocrit , Hct ) , mean red blood volume , and red blood cell distribution width ( RDW ) were measured using a KX - 21 blood cell analyzer ( SYSMEX , Japan ) .






5 . Determination of iron content in rat bone marrow






The fresh bone marrow of the femoral head of the thigh was made into smears , and then stained with acidic potassium ferrocyanide in 24 hours , and the iron staining particles were observed under the microscope for semi - quantitative and qualitative analysis .






6 . Determination of serum iron - regulating protein and erythropoietin in rats






Serum ferritin ( SF ) , serum transferrin receptor ( sTfR ) and serum erythropoietin ( sEPO ) were measured by ELISA . ELISA kit ( available from RD Syste Inc , USA ) : 96 - well microplate , substrate A , substrate B , concentrated wash solution , termination solution , enzyme - linked , standard set . Test instrument : Automatic microplate reader ( Smyth ISE , USA ) .






7 . Statistical methods






The data were analyzed by one - way ANOVA with SPSS 10.0 . The data of experiment was expressed by mean 鹵 standard deviation ( X 鹵 S ) , the significance level was P0.05 , and the significance level was P0.01 .






Results






1 . Effect of psychological stress on serum iron content in rats






Compared with the control group , the SI of the rats in the 3 - day , 7 - day and 14 - day psychological stress group decreased by 28 . 4 % , 27 . 7 % and 34.0 % ( P0.05 ) , and the SI of the rats with the same stress period decreased 28.5 % , 18.3 % and 40.7 % respectively ( P0.05 ) .






2 . Effect of psychological stress on bone marrow iron content in rats






Compared with the control group , the external iron content of bone marrow cells of rats and plantar electric shock rats was significantly decreased in the 7 d group and the plantar electric shock group in 7 d group , and the iron content of bone marrow cells in the rats and the plantar electric shock 14d group decreased further , even absent , while the psychological stress group of the same stress period had no significant difference compared with the sole electric shock group .






3 . Effect of psychological stress on serum iron - regulated protein in rats






Compared with the control group , the SF of the rats with psychological stress increased significantly ( P0.05 ) , while the SF of the rats with continuous psychological stress was 35.9 % and 28 . 3 % ( P0.05 ) , while the sTfR increased 42 . 2 % and 31.5 % ( P0.01 ) , while the changes of serum iron - regulated protein and the psychological stress group were basically consistent with the same stress period ( P0.05 ) .






4 . Effect of psychological stress on erythrocyte parameters in rats






Compared with control group , Hb decreased by 10.0 % and 12.8 % ( P0.05 ) , respectively , and RBC decreased by 4.7 % and 12.9 % ( P0.05 ) .






5 . Effect of psychological stress on food intake and body weight in rats






Compared with the control group , there was no significant change in the feeding amount and weight of the rats with mental stress of 3d , 7d or 14d ( P0.05 ) . The feeding amount and weight of the rats with the same stress period were basically consistent with the changes of the plantar electric shock group ( P0.05 ) .






6 . Effect of psychological stress on liver iron and iron content in rats






Compared with the control group , the content of iron and iron increased by 41.3 % and 24 . 9 % ( P0.05 ) , and the content of iron and iron increased by 12.3 % and 10.4 % respectively ( P0.05 ) .






7 . Effect of psychological stress on erythropoietin in rats






Compared with control group , sEPO decreased by 12.3 % and 35.8 % ( P0.05 ) , and sEPO increased by 26 . 6 % ( P < 0 . 05 ) .






Conclusion






1 . Continuous psychological stress 3d can lead to a marked decrease in serum iron content in rats , while continuous psychological stress 14 d can lead to a further decrease in serum iron content in rats . The changes of serum iron content in rats with psychological stress are basically the same as those of body stress ( plantar electric shock ) .






2 . Psychological stress could significantly decrease the content of iron in rat bone marrow . After 14 days of continuous stress , the decrease of iron level in bone marrow cells of SD rats was more significant than that at 7d . The results showed that the reduction of serum iron level in rats with psychological stress could affect the iron content of rat bone marrow .






3 . Long - term psychological stress ( 7 days of continuous stress ) can lead to a significant decrease in serum iron content in rats , accompanied by a marked increase in serum transferrin receptor levels .






4 . Psychological stress could lead to a significant decrease in RBC , Hb , HCt and so on in SD rats , and the decrease of RDW and RDW increased significantly with the prolongation of the stress period . The results of this study show that the formation of red blood cells can be influenced to some extent due to the decrease of serum iron and bone marrow iron in the psychological stress state for a long time .






5 . The results of this experiment showed that psychological stress had no obvious effect on the consumption and body weight of rats . It suggested that the decrease of serum iron in psychological stress rats could not be related to dietary iron intake . However , it was necessary to further study whether psychological stress could affect dietary iron absorption .






Serum iron ( SI ) and liver and spleen iron contents were measured by atomic absorption spectrophotometer ( Japanese Hitachi Company Z - 2000 ) . 100 u g / mL iron standard stock solution ( GBW08616 ) was provided by the national standard material center .






7 . Continuous stress can increase the content of iron and iron in the liver of rats . It suggests that the redistribution of iron in rats may be one of the causes of the decrease of serum iron content in rats .

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R395

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