【作者】 姜宏；

【导师】 谢俊霞；

【作者基本信息】 青岛大学 ， 生理学， 2004， 博士

【摘要】 帕金森病(PD)的主要病理学改变是黑质(SN)致密带多巴胺(DA)能神经元的进行性丢失，由于铁能够诱发氧化应激反应，因此铁在DA能神经元死亡机制中起着非常关键的作用，研究发现PD病人SN内铁明显增高，但是，目前对脑铁的转运机制知之甚少。二价金属离子转运蛋白1(DMT1)和Ferroportin 1(FP1)的发现为我们阐明PD病人SN内铁聚积的原因带来了一丝曙光。DMT1是1997年发现的哺乳类第一个跨膜铁转运蛋白，它是近年铁代谢研究领域最重大的一项突破。DMT1的mRNA存在两种形式，即“+IRE”和“-IRE”型。它的主要功能是介导小肠铁的吸收和内吞小体铁的移位，目前其在脑内的表达调控机制还不清楚。Ferroportin 1(FP1)是一种跨膜的铁转运蛋白。2000年，三个研究小组才同时分离鉴定了编码该蛋白的基因。FP1的功能主要是将铁从细胞内转运出来。本实验在前期工作的基础上，应用免疫组织化学、高效液相色谱电化学检测技术(HPLC-ECD)观察了1-甲基-4-苯基-1，2，3，6-四氢吡啶(MPTP)制备的PD模型小鼠SN内铁染色和DA能神经元死亡。并应用免疫组织化学方法、免疫荧光标记技术、半定量逆转录聚合酶链反应(RT-PCR)等方法深入探讨MPTP制备的PD模型小鼠和6-羟基多巴胺(6-OHDA)制备的PD模型大鼠SN内铁的增高与DMT1和FP1表达变化的关系。同时还应用免疫组织化学方法研究了DMT1和FP1在成年小鼠脑内的表达，为阐明中枢神经系统铁代谢的机制及铁在PD发病机制中的作用提供实验依据，并为PD的预防和治疗提供新的药物作用靶点。结果表明：中文摘要MPTP腹腔注射3d后，小鼠SN内铁染色即开始增高(P<0.05)，注射MPTP 7d组明显高于3d组(P<0.05)。MPTP腹腔注射3d后，纹状体(Str)内铁染色未观察到变化(P>.05)，7d后纹状体内铁染色明显增高，与正常对照组和3d组相比，差异有统计学意义(P<0，05)。2.两种形式的DMTI在小鼠脑内均有表达，主要分布在大脑皮层、海马、尾壳核、 内侧疆核、丘脑、黑质、弓状核和第三脑室周围的室管膜细胞。在大脑皮层及海 马的CAI和CA3区，DMTI一IRE的表达低于DMTI+J RE，而在内侧疆核和第 三脑室周围的室管膜细胞上，DMTI一IRE的表达却高于DMT卜IRE。3.MPTP组小鼠，SN内酪氨酸轻化酶(TH)免疫阳性细胞数目显著减少，MPTP 处理3d组和7d组分别降低了65.87%和68.16%(P<0 .05)。Str内DA含量也明 显降低，MPTP处理3d组和7d组分别降低了29.83%和54.20%(P<0.05)。4.MPTP组小鼠，SN内“+IRE”型DMTI的表达在MPTP处理后3d开始出现增 高趋势，但与对照组相比差别无统计学意义(P>0.05)。MPTP处理7d后， DMTI+IRE的表达较对照组增高(P<0.05)。而Str内DMTI+IRE的表达在MPTP 处理后3d后即开始增加，与对照组相比差别有统计学意义(P<0.05)，7d后 DMTI+I RE的表达较对照组明显增高，差别有高度统计学意义(P<0.01)。5.MPTP组小鼠，SN内“一IRE”型DMTI表达在各组中都明显增加，与对照组 相比差别有高度统计学意义(P<0.01)。其在Str中的表达变化与SN类似，较对 照组明显增高(P<0.01)。6.MPTP组小鼠，DMTI一IRE在弓状核中的表达较对照组明显增高(P<0 .01)。7.MPTP腹腔注射3d后，DMTI一IRE在小鼠海马的CAI和CA3的表达明显增加(P<0.01)，但7d后表达开始降低，但仍高于对照组(P<0.01)。中文摘要8.MPTP组小鼠，SN内转铁蛋白受体(TfR)的表达没有变化。9.正常小鼠脑内可表达FPI，MPTP制备的PD模型小鼠SN内FPI的表达下降(P<0 .01)。10.免疫荧光结果显示DMTI+I RE主要在大鼠SN细胞的胞浆中表达，且呈不均匀 分布，在胞核内未见表达，而DMTI一IRE既在胞浆表达，也在胞核表达，胞核 中表达量较少。11.6一OHDA制备的PD模型大鼠损毁侧SN中DMTI+I RE和DMTI一IRE蛋白表达 增加(P<0.05)，其mRNA表达水平也增加(P<0.01)。而损毁侧SNT仅mRNA 表达未观察到变化(P>.05)。 通过以上研究，证实新发现的铁转运相关蛋白DMTI和FPI在成年小鼠和大鼠脑内都广泛分布，两种形式的DMTI在脑内的表达差异及在细胞内分布的差异可能与其在铁转运过程中的不同功能有关。MPTP组小鼠SN和Str的DMTI表达增加和SN的FPI表达降低可能是导致SN内铁聚积的主要原因，这种铁的升高可能是由于细胞的胞体和轴突摄铁同时增加所致，SN内铁的聚积可能是导致DA能神经元死亡的主要原因。DMTI一 IRI三还可能参与海马和弓状核的功能，但具体机制不明。在6一OHDA制备的PD模型大鼠，无论从基因表达水平还是蛋白表达水平，同时观察到SN内DMTI表达增加，而T仅的表达不变，说明DMTI的表达增加是SN内铁增高的主要原因。综上所述，DMTI和FPI在脑内的广泛分布，说明它们确实参与脑铁的代谢，对其功能的深入研究对于阐明脑铁代谢的奥秘具有深刻的意义。DMTI在两种PD模型中的表达变化说明DMTI确实参与PD的病理改变，而且它的表达增加可能是导致SN内铁聚积的主要原因，铁的增加进一步引起DA能神经元的死亡，为PD的预防和治疗提供新的药物作用靶点。更多还原

【Abstract】 Iron accumulation in the substantia nigra (SN) has been demonstrated in Parkinson’s disease (PD). Irreversible and regional specific neurodegeneration and the presence of Lewy bodies are the essential pathological hallmarks of PD. The ultimate cause of death of dopamine (DA) neurons in PD is not clear, but a growing body of evidence indicates that increased oxidative stress is one of the main culprits. By its enhancing effect on the generation of oxidative stress in cells, iron might play a key role in the development of PD. Little knowledge is available on how the brain senses and signals that it requires iron or how iron can be removed from the brain. The newly discovered iron transporters, such as divalent metal transporter 1 (DMT1), Ferroportin 1 (FP1), offer a new opportunity for us to understand the accumulation of iron in the PD patients brain. DMT1, identified as the first mammalian transmembrane iron transporter in 1997, is responsible for iron uptake from small intestine and transport from endosomes to cytoplasm. The mRNA of DMT1 has two forms, DMT1+IRE and DMT1-IRE form. The DMT1+IRE form transcript contains a canonical iron-responsive element (IRE) in the 3’ untranslated region, whereas that of DMT 1-IRE form lacks the IRE motif. FP1 is a transmembrane iron transporter that responses for iron efflux from the cell. In 2000, the gene encoding this protein was identified by three groups separately at the same time. However, the exact role of these newly identified iron transport proteins in the brain iron metabolism has not yet been identified. Based on our previous study, using immunohistochemistry and high performance liquid chromatography electrochemical detection (HPLC-ECD), we determine the relationship of elevated iron content and DA cell loss in the SN in l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) treated mice. We also use immunohistochemistry, immunofluorescence and RT-PCR to determine the changed expression of DMT 1 and FP1 in MPTP treated mice and 6-hydroxydopamine (6-OHDA)lesioned rats. Expression patterns of DMT 1 and FPI in the normal mouse and rat brain are also studied. The results were as follows:1. 3 days after MPTP treatment, the iron staining in the mice SN increased significantly (P<0.05), 7 days later, it showed a further increase. While in the striatum (Str), no change was found in iron staining after 3 days of MPTP treatment, however, 7 days after MPTP treatment, it showed apparent increase (P<0.05).2. Two isoforms of DMT1 expressed in the brain of mice. They were,distributed mostly in the cortex, hippocampus, striatum, medial habenular nucleus, thalamus, SN, arcuate nucleus and ependymal cells around the third ventricle. In the CA1 and CA3 region of hippocampus, the expression of DMT 1-IRE was lower than that of DMT1+IRE, while in the medial habenular nucleus and ependymal cells around the third ventricle, the expression of DMT 1 -IRE was much higher than that of DMT1+IRE.3. The number of tyrosine hydroxylase (TH) immunoreactive neurons in the mice SN decreased 65.87% and 68.16% in the group of MPTP 3d and MPTP 7d, respectively. The DA content in the Str also decreased about 29.83% and 54.20%, respectively.4. In MPTP treated mice, the expression of DMT1+IRE in the SN showed no significant increase after 3 days of MPTP treatment, while 7 days later, it showed a significant increase. While in the Str, its expression increased after 3 days of MPTP treatment, a further increase was found after 7 days of MPTP treatment.5. In MPTP treated mice, the expression of DMT 1 -IRE in the SN increased in both two groups, as well as its changed expression in the Str.Abstract6. The expression of DMT 1-IRE in the arcuate nucleus of the mice increased after MPTP treatment (P<0.05).7. The expression of DMT1 -IRE in CA1 and CAS region of hippocampus of the mice increased after 3 days of MPTP treatment, however, after 7 days of MPTP treatment, its expression decreased a little.8. There was no change in the expression of transferrin receptor (TfR) in the SN更多还原