【作者】 张黎；

【导师】 赵正言；

【作者基本信息】 浙江大学 ， 儿科学， 2007， 硕士

【摘要】 缺铁是一常见的儿科疾病，临床研究发现缺铁与情感淡漠、烦躁、焦虑、活动减退，以及认知和注意力减退相联系。尤其是发育早期的缺铁被认为与生长和认知发展延迟密切相关，并且可能对认知行为导致不可逆的损伤。在围产期的幼儿大脑发育中，脑铁降低被证实与髓鞘化不足、多巴胺能的功能损害和神经成熟延迟相关。发育早期缺铁可以产生一系列行为与认知方面的改变，引起机体活性和对环境的反应降低，这些行为在某种程度上与黑质纹状体和边缘系统中的多巴胺功能相关。已有研究显示缺铁可以降低D1和D2受体在尾状核中的密度以及D2受体和多巴胺转运体在依伏神经核中的密度，发育早期缺铁所引起的多巴胺受体及转运体变化并不能为后期补铁所完全纠正。目前，缺铁对行为发育的影响及与脑内多巴胺受体关系的研究，已成为国内外研究的热点之一。多巴胺受体可分为D1类和D2类受体，D1类受体包括D1和D5受体，在脑内主要调控边缘系统的活动，与认知功能有关。D5受体与D1受体的跨膜区相似性达到80％，对多巴胺的亲和力比D1受体高，在脑内主要分布于前额脑锥体细胞层以及海马、基底节区、丘脑等部位。神经药理研究尚未发现区分D1受体和D5受体的特异性药物，D5受体与D1受体各自的功能异同及其意义，一直是令人感兴趣的问题，本实验的目的是观察发育早期缺铁(ID)和无缺铁(CN)大鼠脑内多巴胺D5受体表达及行为的变化，以探讨发育早期缺铁对脑内多巴胺D5受体表达的影响及其与发育和行为改变的可能关系。材料和方法一、材料SD大鼠由浙江大学医学院动物实验中心提供。铁适量膳食(40mg铁／kg，)、铁缺乏膳食(3-5mg铁／kg)均由美国Harlan Teklad公司提供。抗多巴胺D5受体单克隆抗体由美国CHEMICON公司提供。二、方法1、建立动物模型16只SD成年雌鼠接受铁适量饮食1周调整体内铁状态后配种，以发现阴道栓为孕0天，随机分为两组，缺铁组(ID组)(n=8)接受铁缺乏饮食(3-5mg铁／kg)，对照组(CN组)(n=8)接受铁适量饮食(40mg铁／kg，)。怀孕3周后雌鼠娩出仔鼠(缺铁组n=70，对照组n=85)，以初生当天记为出生后0天(P0)。仔鼠生后满21天起断乳，分别喂饲与母鼠相同的饮食，直至生后25天。25天后，两组仔鼠均喂饲铁适量饮食。母鼠在孕0、孕20天鼠尾采血，测定血红蛋白(Hb)、血球压积(Hct)等血液学指标以评估血铁水平。2、行为功能评估于生后6、9、12、15、18、21天进行发育里程碑评估(包括前肢悬挂试验、翻身校正能力试验，反趋地性反应试验)，生后24，29天进行触须前肢放置试验测试感觉运动功能，生后15、25、35天进行旷场试验测试情绪认知相关行为。3、脑冰冻切片免疫荧光染色-荧光显微镜扫描检测于仔鼠出生后15、25、35天，行全脑制备冰冻切片进行免疫荧光染色后，荧光显微镜下观察扫描及照相，观察缺铁组和对照组多巴胺D5受体表达情况。4、统计学分析数据以均数±标准差(mean±SD)表示，采用SPSS／PC+统计软件，不同组间数据比较采用独立样本t检验，组内各日龄间采用One-Way ANOVA方法进行比较。结果1、动物模型建立结果(1)两组母鼠监测体重均无显著性差异(P＞0.05)。缺铁组母鼠孕20天血红蛋白(Hb)及血球压积(Hct)值与自身孕0天及对照组比较均有显著降低(P＜0.001)。(2)缺铁组仔鼠体重增加慢，体形小，行动迟缓，反应迟钝，张眼晚，毛发萌出晚且稀疏；两组仔鼠体重在同日龄比较均有显著性差异(P＜0.05)。2、行为功能评估结果(1)缺铁组仔鼠与同日龄对照组比较悬吊时间明显缩短，两者具有显著性差异(P＜0.05)，缺铁组仔鼠与同日龄对照组比较翻身校正时间和反趋地性反应所用时间与对照组比较均有显著性差异(P＜0.05)。(2)出生后24、29天，缺铁组仔鼠与同日龄对照组(CN)比较两侧触须前肢放置反应均有显著性差异(P＜0.05)。(3)生后15天，缺铁组(ID)仔鼠垂直勘探次数与理毛次数明显小于对照组(P＜0.05)；生后25天，缺铁组仔鼠水平活动格数、垂直勘探次数及理毛次数均明显小于对照组(P＜0.001)；生后35天，缺铁组仔鼠水平活动格数小于对照组(P＜0.05)3、免疫荧光研究结果(1)仔鼠生后15、25、35天，缺铁组仔鼠基底节区多巴胺D5体阳性表达明显低于对照组(P＜0.001)。(2)缺铁组仔鼠生后25天D5受体阳性表达显著高于同组15日龄(P＜0.001)。生后35天阳性表达要明显高于同组25日龄(P＜0.05)。(3)对照组仔鼠生后25天的D5受体阳性表达要显著高于同组15日龄(P＜0.05)，而与同组35日龄比较无显著性差异(P＞0.05)。结论1、围产期缺铁的母鼠所生仔鼠可以表现出明显的发育迟滞，表现为体重增长缓慢，生理发育指标明显落后，行动迟缓，反应迟钝。2、发育早期缺铁对于仔鼠的一般运动协调能力、反射发育、感觉运动功能，情绪认知活动都有不同程度的损伤，而一部分感觉运动功能和情绪认知活动改变在一定时间的后期补铁中不能被完全纠正。3、多巴胺D5受体在脑内的发育表达遵循一定的时间变化规律。4、发育早期缺铁仔鼠脑内多巴胺D5受体表达下降，脑基底节区多巴胺D5受体表达峰值出现延后。一定时间的补铁不能使脑基底节区多巴胺D5受体的表达恢复正常。更多还原

【Abstract】 Iron deficiency (ID) is a common disorder in pediatric patients. Clinical studies demonstrate that iron deficiency is associated with apathy, irritability, anxiety, hypoactivity, and decreased cognition and attention. Iron deficiency in early development is associated with retardation in growth and cognitive development, and the effects on cognition may be irreversible.Iron plays an important role in the perinatal brain development. Brain iron deficiency in early development has multiple consequences in neurobiology including hypomyelination, impared dopaminergic function and delayed neuromaturatian. Iron deficiency in early life alters the course of behavioral and cognitive development in humans, leading to decreased physical activity and responsiveness to the environment. These behavioral changes are partly related to dopaminergic function in nigrostriatal and mesocorticolimbic pathways. Earlier studies show that iron deficiency decreased densities of D1 and D2 receptors in the caudate-putamen, as well as D2 receptor and dopamine transporter (DAT) densities in the nucleus accumbens. The changes in DA biology induced by perinatal iron deficiency are not completely normalized despite replenishment of iron status later.The effects of iron deficiency on behavior and dopamine receptor have been widely investigated in humans and in experimental animals.The dopamine receptor subtypes are divided into two major subclasses: the D1-like and D2-like receptors, The D1-like family (D1 and D5 receptors) has been involved in the regulation of motor activity at limbic system and is related to cognition. The D1 and D5 receptors share a 80% identity in their transmembrane domains. DA has -10 times higher affinity for the D5 receptor than the D1 receptor. D5 receptor can be found in rostral forebrain cortex, hippocampus, striatum and substantia nigra as well as thalamus. So far, it has not been possible to pharmacologically differentiate D1 and D5 receptors. There is still a question about distinct roles of D1 and D5 dopamine receptors in their function and clinical manifestation. This study was designed to investigate the effects of early Iron deficiency on dopamine D5 receptor expression, to determine whether Iron deficiency in early development produces behavioral changes not reversible with subsequent iron repletion, and to examine the relation between D5 receptor expression and behavioral changes.Materials and MethodMaterialsSprague-Dawley (SD) rats were obtained from the Center for Experimental Animal Center of Zhejiang University school of medicine. Iron sufficient diet (40mg iron/kg) and iron deficient diet (3-6mg iron/kg) were purchased from Harlan Teklad(USA), Madison, WI. The mouse anti-dopamine D5 receptor monoclonal antibody was purchased from CHEMICON International, Inc. USA.Method1、 Establishment of Animal ModelsBefore mating, 16 SD female rats were treated with an iron sufficient diet for a week to regulate iron status. Then female rats were randomly assigned to two experimental diet groups. Iron deficient group (ID) (n=8) received an iron deficient diet (3-6mg iron/kg) and the control group (CN) (n=8) received an iron sufficient diet (40mg iron/kg). ID (n=70) and CN (n=85) pups were weaned at 21 days of age (P21) and fed the same diet as their dams. All the pups were treated with an iron sufficient diet at 25 days of age (P25). Blood samples of dams for hemoglobin and hematocrit analysis were obtained by thigh venous puncture.2、 Behavioral AssessmentsThe level of development was assessed and recorded at days P6, P9, P12, P15, P18 and P21. The test for development assessment included forelimb hanging test, surface righting reflex and negative geotaxis reflex. Upper extremity sensorimotor function was assessed with vibrissae-evoked forelimb placing test, which is sensitive to nigrostriatal dopamine tract impairments, at P24 and P29. At P15, P25 and P35, pups received the open field test.3、 Immunofluorescence assay (IFA)Whole brain of pups was removal and stored to take 8um frozen section for immunofluorescent staining at P15, P25 and P35. Dopamine D5 receptor expression levels in the two groups were evaluated under the fluorescene microscope.4、 Statistical analysisAll values are expressed as mean ± SD. All the data were evaluated by theSPSS/PC+ software package. Significance of differences between groups were calculated using unpaired T-test. Difference between age in the same group was analyzed by one-way ANOVA test. P<0.05 was considered as statistically significant.Results1、 Results of Animal Models(1) No differences was observed in the body weight of dams between the two groups (P>0.05). The ID dams showed a significant decrease in the hemoglobin and hematocrit levels at 20 days of gestation (G20) than GO; the level also decreased compared to that of the CN dams (P<0.001).(2) As the pups aged, the weights of ID pups were lower than that recorded for age-matched CN pups (P<0.05).2、 Behavior(1) ID pups performed worse than age-matched CN pups in the development assessment tests, including forelimb hanging test, surface righting reflex and negative geotaxis reflex (P<0.05).(2) ID pups had reduced bilateral vibrissae-evoked forelimb placing after weaning as compared to age-matched CN pups. (P<0.05)o(3) ID pups at P15 and P25 showed significantly decreased levels of standing exploration and spontaneous groom in a new environment than age-matched controls (P<0.05). Total distance traveled by pups was also significantly lower in ID pups at P25 (P<0.001). At P35, total distance traveled by ID pups was still significantly lower than that by age-matched CN (P<0.05).3、 Immunofluorescence assay(1) Dopamine D5 receptor expression in the basal ganglia was significantly lower in ID pups compared to that in age-matched CN pups at P15, P25 and P35.(2) D5 receptor expression for ID pups was significantly higher at P25 than P15 (P<0.001). ID pups at P35 showed an increase of D5 receptor expression compared to the pups at P25 (P<0.05).(3) D5 receptor expression for CN pups was significantly higher at P25 than P15 (P<0.05). CN pups at P35 showed no difference in D5 receptor expression compared to the pups at P25 (P>0.05).Conclusions1、 The pups from perinatal iron-deficient dams showed a significantly retardation ingrowth and development.2、 Iron deficiency in early development of the pups lead to more or less impairmentsincluding regulation of motor coordination, reflect development, sensorimotorfunction, emotion and cognition. And part of the changes in sensorimotor functionand cognition that vary with duration of perinatal iron deficiency are not completelynormalized despite replenishment of iron status.3、 Dopamine D5 receptor expression follows a time-varying course of developmentin the rat brain.4、 Iron deficiency in early development reduced the expression of dopamine D5receptor in rat brain. D5 receptor expression in basal ganglia peaked later with adelay the pups with iron deficiency in early development. The changes in D5receptor expression that vary with duration of early iron deficiency are notcompletely normalized despite replenishment of iron status.更多还原