心理应激对大鼠造血系统铁代谢的影响

发布时间：2018-04-12 12:54

本文选题：心理应激 + 血清铁　；参考：《第二军医大学》2007年硕士论文

【摘要】： 铁是合成血红蛋白的重要原料。大量研究资料表明血清铁含量下降可影响血红蛋白的合成,进而影响红细胞的形成与成熟,甚至引起缺铁性贫血[1,2]。据WHO报道,全世界患缺铁性贫血人数约有10亿。我国缺铁性贫血的发病率也很高,孙建琴等报道上海儿童、青少年人群高达19.9%[3]。由于缺铁性贫血可影响人体多种生理功能和生殖能力[4,5],因此该病的防治一直受到人们极大的关注。引发缺铁性贫血的主要原因是铁摄入不足和铁丢失过多[7]。但也有研究发现,部分人群即使在膳食铁供给充足且无过量丢失的情况下,缺铁性贫血的发病率依然居高不下[8];也就是说缺铁性贫血的高发可能存在膳食铁摄入量以外的影响因素。但迄今为止,研究者对造成高膳食铁摄入量和高缺铁性贫血发生率这一矛盾现象的原因还不清楚[9-11]。我们在以往研究中发现,应激可以导致血清铁大幅度下降,例如:加速度刺激可导致人体血清铁下降52.3%[12];热暴露可引起大鼠血清铁下降31%[13]。国外也有不少同类研究报道,例如:Navas FJ等报道大运动量训练可引起运动员血清铁下降[14];Smith SM等发现航天飞行可导致宇航员血清铁下降[15];Nikolova- Todorova Z等提出手术创伤也可造成病人血清铁下降[16]。但迄今为止,尚无关于心理应激是否会导致血清铁下降,以及心理应激引起的血清铁下降是否会影响红细胞生成的报道。本研究对不同心理应激周期SD大鼠血清铁、铁调节蛋白及红细胞生成的变化进行了观察。目的了解心理应激对大鼠造血系统铁代谢及红细胞生成的影响,为阐明心理应激与红细胞代谢关系提供实验基础。方法 1.实验动物分组雄性SD大鼠,体质量80±5g,上海西普尔-必凯公司提供。单笼饲养,自由饮食,室温24±1℃,相对湿度50～60%,12h/12h光暗周期节律。适应3d后按体重随机分成空白对照组(control group, CG)、心理应激组(psychological stress group, PSG)和足底电击组(foot-shock stress group, FSG),每组再分为3d、7d和14d三个亚组。 2.建立SD大鼠心理应激模型按照文献[17]采用Communication Box System制作大鼠心理应激模型。Communication Box System由透明丙烯酸板组成,一半小室(A)底部铺板绝缘,另一半小室(B室)通电。在B室的实验大鼠接受30min/d,足底电击(电压90V,电流0.80mA);电击组大鼠接受电击时跳跃,尖叫,在A室的实验大鼠通过视觉听觉产生恐惧的心理反应,即为心理应激大鼠模型。按计划完成不同周期应激暴露后,各组大鼠立刻断头处死取组织标本备用。 3.大鼠血清、肝脏和脾脏铁含量的测定采用原子吸收分光光度计(日本日立公司Z-2000型)火焰法测定血清铁(serum iron, SI)和肝脾铁含量。100 u g/mL铁标准贮备液(GBW08616)由国家标准物质中心提供。 4.大鼠红细胞参数的测定使用KX-21血细胞分析仪(SYSMEX,日本)测定血红蛋白(hemoglobin,Hb)浓度、红细胞计数(red blood cell count ,RBC)、红细胞压积(hematocrit, Hct)、平均红细胞体积(mean corpuscular volume,MCV)和红细胞分布宽度(red blood cell distribution width,RDW)。 5.大鼠骨髓铁含量的测定取大腿股骨头新鲜骨髓制成涂片,于24h内用酸性亚铁氰化钾进行染色,显微镜下观察铁染色颗粒,进行半定量和定性分析。 6.大鼠血清铁调节蛋白及促红细胞生成素含量的测定应用ELISA方法测定大鼠血清铁蛋白(serum ferritin, SF)、血清转铁蛋白受体(serum transferring receptor, sTfR)和血清促红细胞生成素(serum erythropoietin, sEPO)。ELISA试剂盒(购自RD Sysytems Inc,USA)组成:96孔酶标板、底物A、底物B、浓缩洗涤液、终止液、酶联物、标准品一套。测试仪器:自动酶标仪(SUNRISE,美国)。 7.统计方法实验数据采用SPSS10.0统计软件包采用one-way ANOVA检验进行数据分析,实验数据以平均数±标准差( X±S)表示,显著性水平为P0.05,非常显著性水平为P0.01。结果 1.心理应激对大鼠血清铁含量的影响与对照组相比,3d、7d和14d心理应激组大鼠的SI分别下降28.4%、27.7%和34.0%(P0.05),3d、7d和14d足底电击组大鼠的SI则分别下降28.5%、18.3%和40.7%,(P0.05)。相同应激周期的心理应激组与足底电击组大鼠相比,SI含量无显著差异(P0.05)。 2.心理应激对大鼠骨髓铁含量的影响酸性亚铁氰化钾染色显示,与对照组比较,心理应激7d组大鼠和足底电击7d组大鼠骨髓细胞外铁含量明显减少,心理应激14d组大鼠和足底电击14d组大鼠骨髓细胞外铁含量进一步下降,甚至缺如;而相同应激周期的心理应激组与足底电击组相比,无显著差异。 3.心理应激对大鼠血清铁调节蛋白的影响与对照组相比,心理应激3d大鼠的SF显著升高(P0.05);而连续心理应激7d和14d组大鼠的SF分别下降35.9%和28.3%(P0.05),同时伴有sTfR分别升高42.2%和31.5%(P0.01)。而相同应激周期的足底电击组大鼠血清铁调节蛋白的变化和心理应激组基本一致(P0.05)。 4.心理应激对大鼠红细胞参数的影响与对照组相比,7d和14d心理应激组大鼠的Hb分别下降10.0%和12.8%(P0.05)、RBC分别下降4.7%和9.8%(P0.05)、Hct分别下降7.0%和12.9%(P0.05)、MCV分别下降1.6%和7.3%(P0.05),而RDW则分别增加11.2%和22.5%(P0.05);7d和14d足底电击组大鼠的Hb分别下降8.7%和9.9%(P0.05)、RBC分别下降6.1%和10.5%(P0.05)、Hct分别下降5.4%和11.4%(P0.05)、MCV分别下降3.6%和1.5%(P0.05),而RDW则分别增加3.6%和6.1%(P0.05)。相同应激周期的心理应激组大鼠红细胞各参数和足底电击组大鼠相比,均无显著差异(P0.05)。 5.心理应激对大鼠进食量和体重的影响与对照组相比,心理应激3d、7d或14d的大鼠进食量和体重均无明显变化(P0.05)。相同应激周期的心理应激组大鼠进食量和体重与足底电击组变化基本一致(P0.05)。 6.心理应激对大鼠肝铁和脾铁含量的影响与对照组相比,心理应激7d和14d组大鼠肝铁含量分别升高41.3%和24.9% (P0.05),脾铁含量分别升高12.3%和10.4%(P0.05);足底电击7d和14d组大鼠肝铁含量分别升高24.7%和10.3% (P0.05),脾铁含量分别升高15.1%和17.8%(P0.05)。相同应激周期的心理应激组大鼠肝、脾铁含量和足底电击组大鼠相比,无显著差异(P0.05)。 7.心理应激对大鼠促红细胞生成素的影响与对照组相比,14d时心理应激组和足底电击组sEPO分别下降12.3%和35.8%(P0.05); 14d足底电击组与心理应激组相比,sEPO显著升高26.6%(P0.05)。结论 1.连续心理应激3d可导致大鼠的血清铁含量明显下降,而连续心理应激14d可导致大鼠血清铁含量进一步下降。心理应激大鼠血清铁含量的变化趋势与躯体应激(足底电击)大鼠基本一样。 2.心理应激可以导致大鼠骨髓铁含量显著降低。连续应激14d时,SD大鼠骨髓细胞外铁染色减少程度较应激7d时更显著。上述结果表明,心理应激大鼠血清铁水平降低可进而影响其骨髓铁含量。 3.较长时间的心理应激(连续应激7d)可导致大鼠血清铁蛋白含量显著下降,同时伴有血清转铁蛋白受体含量显著升高。综合心理应激可影响血清铁和骨髓铁含量等观察结果,提示心理应激引起的血清铁含量降低,可导致机体贮存铁和骨髓铁减少,进而影响到骨髓的红细胞生成。至于应激3d组大鼠SF含量增加,可能与SF为急性时相反应蛋白有关,即SF在应激早期升高;但随着应激状态的延续,血清铁持续降低,SF含量逐渐下降。确切原因有待进一步探讨。 4.心理应激可导致SD大鼠的RBC、Hb、HCt等参数明显下降,下降幅度随着应激周期的延长而增加;同时伴有MCV显著下降,RDW显著升高,RDW/MCV显著升高(RDW/MCV比值升高,在临床中作为小细胞低色素性贫血的诊断指标之一)。综合本课题研究结果表明,长时间处于心理应激状态,可因为血清铁和骨髓铁的降低而在一定程度上影响红细胞的生成。 5.本实验结果显示,心理应激对大鼠进食量和体重均无明显影响,提示心理应激大鼠血清铁降低可能与饮食铁摄入量无关。然而,心理应激是否会影响饮食铁吸收率则需要进一步研究。 6.一般情况下,红细胞生成绝对不足或相对不足可导致sEPO含量升高,本实验中心理应激组大鼠在连续应激14d时sEPO却显著降低。心理应激导致sEPO下降的原因,以及心理应激导致的sEPO降低是否为影响红细胞生成的原因之一,均有待于进一步探讨。 7.连续应激可造成大鼠肝铁和脾铁含量显著升高。提示心理应激条件下,大鼠体内铁的重新分布可能是其血清铁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 卤 standard deviation ( X 卤 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 .

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R395

【参考文献】