视网膜铁代谢与年龄相关性黄斑变性实验研究

发布时间：2018-08-07 06:58

【摘要】：年龄相关性黄斑变性（age related macular degeneration, AMD)是目前影响老年人视功能主要疾病之一，氧化应激是年龄相关性黄斑变性重要发病机制，活性氧簇可以促进视网膜变性的病理改变，铁可以通过芬顿反应产生活性氧簇，过量的铁可以诱导氧化应激损伤。铁代谢异常通过诱导氧化应激损伤可能参与了年龄相关性黄斑变性的发生发展。铁调节蛋白(hepcidin，Hepc)是铁代谢调控过程中的重要因子，它可以通过与膜铁转运蛋白1（ferroportin，Fpn）相结合并使后者发生降解，由于Fpn是目前唯一已知的细胞排出铁的通路，因此，对铁调节蛋白的精细调控对维持细胞内铁稳态具有重要的意义。本课题研究旨在探讨铁调节蛋白在视网膜局部所受的调控机制和铁螯合剂对铁过载视网膜的保护作用。为探讨铁调节蛋白的调控机制，本课题采用了骨形态发生蛋白-6（bonemorphogenetic protein6，Bmp6）基因敲除小鼠，Hepc基因肝脏特异性敲除小鼠，铁蔗糖注射剂（iron sucrose injection，Venofer）注射小鼠模型，分别取不同月龄小鼠，通过免疫荧光，real-time PCR等技术检测形态学及生物化学等指标，从而探讨铁调节蛋白在视网膜局部的调控机制。实验结果显示Bmp6基因敲除型小鼠视网膜铁水平随年龄增高而逐渐积聚，并显著高于对照组，Hepc的表达并未受Bmp6敲除的影响，在视网膜铁水平升高的同时也随之升高；Hepc基因肝脏特异性敲除小鼠视网膜的铁水平也随着年龄逐渐增高，并显著高于对照组；给予铁剂的C57BL/6J小鼠的视网膜色素上皮（retinal pigment epithelium，RPE）发生了铁过载，局部铁调节蛋白表达虽然发生了变化，但未能对抗全身铁水平的变化及减少视网膜铁吸收。综上，我们得出结论铁调节蛋白在视网膜局部发挥功能，其调控不受Bmp6通路影响，循环中的铁水平是铁调节蛋白的强调节因子。为探讨铁鳌合剂去铁酮(deferiprone，DFP)的视网膜保护作用，本课题采用了Hepc基因敲除小鼠模型，给予DFP干预12个月后处死，，使用视网膜电图描记术（ERG），眼底照相，免疫荧光，real-time PCR等技术检测视网膜功能学、形态学及分子生物学指标。结果显示使用DFP后，不仅视网膜的功能较对照组相比损害较轻，视网膜形态学也证实光感受器细胞存活较多，RPE细胞的自发荧光及肥大细胞数量和程度较轻，分子生物学结果显示视紫红质（rhodopsin，Rho）及RPE65等基因表达水平显著高于对照组。实验结果提示DFP可以显著降低视网膜铁水平，并降低氧化应激水平，保护视网膜功能。
[Abstract]:Age-related macular degeneration (age related macular degeneration, AMD) is one of the main diseases affecting the visual function of the elderly. Oxidative stress is an important pathogenesis of age-related macular degeneration. Reactive oxygen species cluster can promote the pathological changes of retinal degeneration. Iron can produce reactive oxygen species by Fenton reaction, and excessive iron can induce oxidative stress damage. Abnormal iron metabolism may be involved in the occurrence and development of age-related macular degeneration by inducing oxidative stress damage. Iron regulatory protein (hepcidin Hepc) is an important factor in the regulation of iron metabolism. It can be combined with membrane iron transporter 1 (FPN) and degrade the latter. Because Fpn is the only known pathway of iron excretion, so, The fine regulation of iron regulatory proteins plays an important role in maintaining the homeostasis of iron in cells. The aim of this study was to investigate the regulatory mechanism of iron regulatory proteins in the retina and the protective effect of iron chelating agents on iron overload retina. In order to investigate the regulatory mechanism of iron regulatory protein, the bonemorphogenetic protein 6 (bonemorphogenetic protein 6 Bmp6) gene knockout mice were used in this study. The liver specific knockout mice of Hepc gene were injected with iron sucrose injection (iron sucrose injection Venofer. Morphological and biochemical indexes were detected by real-time PCR, so as to explore the regulatory mechanism of iron regulatory proteins in the retina. The results showed that the retinal iron level of Bmp6 knockout mice gradually accumulated with age, and the expression of HEC was not affected by Bmp6 knockout, and increased with the increase of retinal iron level. The iron level of the retina of the Hepc liver-specific knockout mice increased with age and was significantly higher than that of the control group. Iron overload occurred in the retinal pigment epithelium (retinal pigment epithelium of the C57BL/6J mice treated with iron. Although the expression of local iron regulatory protein changed, it could not resist the change of iron level and decrease the absorption of iron in retina. In conclusion, iron regulatory proteins play a role in the local retina, and their regulation is not affected by the Bmp6 pathway. The iron level in the circulation is an important factor of iron regulatory proteins. In order to investigate the protective effect of deferriferone on the retina, the Hepc gene knockout mice model was used in this study. After 12 months of DFP intervention, the mice were killed, and (ERG), fundus photography was performed by electroretinography. Retinal function, morphology and molecular biological indexes were detected by real-time PCR. The results showed that not only the function of the retina was less damaged than that of the control group, but also the number and degree of autofluorescence and mast cells in the photoreceptor cells were less than those in the control group. The results of molecular biology showed that the expression levels of rhodopsin Rho and RPE65 were significantly higher than those of the control group. The results suggest that DFP can significantly reduce the level of iron in the retina, reduce the level of oxidative stress and protect the retinal function.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R774.1

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