【作者】 付丽娟；

【导师】 常彦忠； 段相林；

【作者基本信息】 河北师范大学 ， 动物学， 2005， 硕士

【摘要】 铁是机体必需的营养元素。今天人们已经认识到许多疾病与铁代谢紊乱有关。随着世界人口老龄化的加剧,在这些铁代谢紊乱相关疾病中,神经退行性疾病越来越引起人们的关注。目前,脑铁代谢已成为一个非常热门的研究领域。 铁调素hepcidin为一种富含半胱氨酸的抗菌多肽。它对铁稳态的调控作用是由Pigeon等首先发现的。后来通过基因敲除和转基因小鼠的研究进一步得到其调控机体铁平衡的直接证据。由此,学者们认为铁调素很可能就是长期以来人们一直在寻找的存在于小肠、肝脏、骨髓和巨噬细胞之间的可溶性信号物质。它可能通过调节DMT1、FP1等铁转运蛋白的表达,抑制十二指肠对铁的吸收及巨噬细胞的铁释放。然而,铁调素是否在脑内有表达,以及对脑铁代谢是否有调控作用至今未见报道。鉴于此,本论文对脑内hepcidin进行鉴定,并从在体和离体两个方面研究铁调素在脑铁代谢中的作用,实验结果显示: 1.小鼠脑内有hepcidin基因表达 提取小鼠脑脉络丛、大脑皮层、间脑及中脑、小脑和脑桥与延髓组织总RNA进行RT-PCR表明,大脑皮层、间脑和中脑、小脑、脑桥与延髓、脉络丛均有hepcidin表达,其中以脉络丛表达较高,但与肝脏相比,表达量较低。回收PCR产物并测序,将结果与小鼠肌肉抗菌多肽hepcidin mRNA(cDNA clone MGC:35592 IMAGE:5097807)序列比对,测出的137 bp一致性为100%,证明PCR产物即为小鼠hepcidin。 2.侧脑室微量注射hepcidin,导致不同脑区DMT1、FP1蛋白表达的不同变化 小鼠侧脑室微量注射hepcidin 12 h后,大脑皮层、纹状体DMT1(+IRE)表达与注射生理盐水组相比显著升高(P<0.01);DMT1(-IRE)、FP1表达与注射生理盐水组相比显著降低(P<0.01)。在黑质,DMT1(+IRE)表达与注射生理盐水组相比显著降低(P<0.01);DMT1(-IRE)、FP1表达较注射生理盐水组显著升高,且差异极显著(P<0.01)。侧脑室及第三脑室脉络丛两种DMT1表达较注射生理盐水组显著升高,且差异极显著(P<0.01),FP1表达较注射生理盐水组降低,但差异不显著。 3.成功对大脑皮层和海马原代神经元进行体外培养更多还原

【Abstract】 Iron is an essential element that is required for growth and survival. Nowadays, people have realized that many human diseases are associated with iron metabolism disorders. With the increase of old population, neurodegenerative diseases are paid attention gradually among these disorders related with iron. Recently, the brain iron metabolism research becomes very hot.Hepcidin was found to be a small cysteine-rich peptide with intrinsic antimicrobial activity. The first link between hepcidin and iron metabolism come from the study of Pigeon et al. The direct proof that hepcidin can modulate iron balance of the body was resulted from the observation of the mice whose hepcidin gene were knocked out and overexpressed. Therefore, hepcidin appears a key soluble signal among gut, liver, bone marrow and reticuloendothelial system, which was the long-postulated iron regulator. Obviously, plasma hepcidin inhibits iron uptake in the duodenum and iron release from macrophages through modulating the expression of DMTl and FP1. However, there is not a report on the expression of hepcidin in the brain up to now. Likewise, whether it can regulate brain iron metabolism has not to be answered. Therefor, in the dissertation, the expression of hepcidin in mice brain was identified and its role in the brain iron metabolism was investigated in vitro and in vivo. The results showed:1. Hepcidin mRNA was expressed in the mice brain.Total RNA was extracted from choroid plexus, cerebral cortex, diencephalon and mesencephalon, cerebellum, medulla oblongata and pons in mice. RT-PCR analysis demonstrated that hepcidin mRNA was expressed in all areas of mouse brain, in choroid plexus the mRNA expression was highest, although its level is generally lower than that of liver. The PCR products were purified and verified the sequence. Sequence analysis of the 137 bp fragment revealed the presence of 100% identity, compared with mouse muscles hepcidin antimicrobial peptide mRNA （cDNA clone MGC: 35592 IMAGE: 5097807）. The result exhibited that the amplified products is hepcidin cDNA.2. Effect of hepcidin microinjection into the lateral cerebral ventricle on DMTl, FP1protein synthesis in different brain regions.After hepcidin was injected into the lateral cerebral ventricle for 12 h, the expression of DMT1 （+IRE） increased significantly （P<0.01）; the expression of DMT 1 （-IRE） and FP1 decreased significantly （P<0.01） in cerebral cortex and in corpus striatum. But in substantia nigra, the DMT1 （+IRE） protein synthesis was significantly decreased （P<0.01）; the DMT1 （-IRE） and FP1 protein synthesis was significantly increased （P<0.01） respectively. In choriod plexus of the lateral cerebral ventricle and the third ventricle, hepcidin induced the expression of DMT1 both （+IRE） and （-IRE） significantly higher （PO.01） than that of the control, but there was no significant change between two groups in the expression of FP1.3. In vitro, the cerebral cortex and hippocampus neurons were cultured successfully.Isolated neurons from the cerebral cortex and hippocampus of newborn SD rats were cultured for 7 days. The cell concentrition was approximate 2×l0⁶ each chamber. After identified by neuron-specific enolase, the purity of the cultural neurons was more than 95%. Then cells were used to experiment.4. Effect of hepcidin on iron uptake of cultured neurons.The iron absorption of neurons was elevated with the increase of the iron concentrition from 0 to 2 μM. When the iron concentrition was higher than 2μM, the level of cells uptake iron was unchangeable. Thus, we selected 2 μM to detect the ability of neurons iron uptake. Hepcidin （25μg/ml） was added in the media for 24 h, the cells were used to take up ⁵⁵Fe. The amount of ⁵⁵Fe in the hepcidin treated group cells was great higher than that of the control （P<0.05）. Conclusions1. In the present study, hepcidin was identified in the mice brain by RT-PCR. The data showed that all 5 regions which were examined have the ability to express hepcidin mRNA, although the level is generally lower than that of liver. The futher investigation should be studied using in situ hybridization.2. After hepcidin injected into the lateral cerebral ventricle 12 h, the expression of DMT1, FP1 was significantly changed in cerebral cortex, corpus striatum, substantia nigra and choriod plexus. This result revealed hepcidin could modulate iron-transport molecules and regulate iron balance in the mouse. It is obvious that hepcidin appears specifically regional regulation. Because the mechanism of brain iron homeostasis is much more elaborate, the regulation of brain iron metabolism by hepcidin might be more complicated than other更多还原