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视网膜内Kir通道的分布、表达和发育
Expression、Localization and Development of Kir Channels in Retina
【作者】 潘爱华；
【导师】 张阳德；
【作者基本信息】 中南大学 ， 外科学， 2004， 博士
【摘要】 目的：本研究在mRNA和蛋白水平探讨在牛视网膜内Müller细胞的Kir2.1和Kir4.1的表达和分布及其功能，Kir7.1在牛视网膜色素上皮细胞(RPE)的表达和分布；同时研究三种Kir通道在不同种属大鼠、猴和人的视网膜的分布及在大鼠视网膜中发育情况。 方法：从牛神经视网膜和视网膜色素上皮分离mRNA，用RT-PCR，和Northern Blot斑点杂交分析不同亚型Kir2.1、Kir4.1和Kir7.1在牛神经视网膜和RPE的mRNA的表达；从牛神经视网膜和视网膜色素上皮分离蛋白质，用Western Blot斑点杂交分析检测不同亚型Kir蛋白Kir2.1、Kir4.1和Kir7.1在牛神经视网膜和RPE的分布；用间接免疫荧光组织化学和ABC法免疫组织化学在冰冻切片上检测三种Kir蛋白在牛、大鼠、猴和人视网膜中的定位及在不同年龄大鼠视网膜中的发育情况。 结果：RT-PCR结果显示Kir2.1 cDNA产物在神经视网膜中强表达，在RPE中弱表达；Kir4.1 cDNA产物仅在神经视网膜中表达，RPE内无表达；Kir7.1 cDNA产物在RPE中强表达，在牛神经视网膜弱表达。Northern斑点杂交分析显示Kir2.1 cDNA探针杂交在牛神经视网膜中检测到6.5KB大小的带，Kir4.1 cDNA探针杂交在神经视网膜中检测到5.5KB大小的带，两者均在RPE中均无表达；Kir7.1 cDNA探针杂交在牛神经视网膜和RPE中均得到大约1.5KB的带，且RPE中的mRNA表达量显著多于神经视网膜。Western斑点杂交在牛神经视网膜中得到约60 Kda单体的Kir2.1和约60 Kda单体的Kir4.1；在RPE仅检测约53Kda单体的Kir7.1，这些蛋白都能被相应产生抗体的多肤所抑制。间接免疫荧光组织化学显示，Kir2.1蛋白主要分布在Muller细胞与神经元相接触的细胞膜区域;与Muller细胞的特异性标记蛋白质抗谷氨酸合成酶抗体(GS)双重标记显示其分布特性重叠，在RPE未检测K 1 r2.1蛋白的免疫活性存在;Kir4.1蛋白集中分布于Muller细胞的内侧突起，与GS蛋白双重标记显示其分布特性重叠，RPE未检测Kir4.l蛋白的免疫活性存在;Kir7.1主要分布于RPE游离面及其突起的全长，与特异性标记RPE突起的Ezrin蛋白完全重叠，Na+一K+ATP酶在不同部位的RPE表达不同，Na+一K+AfP高表达的RPE细胞与Kir7.1的分布特性重叠。ABC免疫组织化学显示Kir2.1和Kir4.1分布在猴和人视网膜M沮ler细胞的不同部位，而Kir7.1分布于人和猴的RPE细胞的游离面及其突起。在不同年龄的大鼠中，KirZ.l和Kir4.1均在P，6表达接近成年水平，Kir7.l在P:表达接近成年水平。 结论:这些结果显示，KirZ .1和Kir4.1在P16接近成年水平，分布于视网膜Mulle:细胞的不同部位，在不同种类之间无明显差别，这种分布特性可能与完成Muller细胞功能一保持细胞外K+的平衡，促进K+的内流有重要关系。Kir7.1在P7表达接近成年水平，分布于RPE的游离面和突起的全长，在不同种类之间无明显差别，说明Kir7.1的特性可能促进K+的转运和光引起的视网膜下间隙K+浓度的RPE电反应相关。
【Abstract】 Purpose: The purpose of this study was to detect expression and localization of kir2.1 and kir4.1 in bovine retina, identify the molecular basis of the apical membrane K+ conductance in native bovine retinal pigment epithelium (RPE), study localization of Kir channels in different species and development of Kir channels in murine retinae.Methods. RT-PCR, Northern blot, and Western blot analyses were used to detect the expression of the inwardly rectifying K+ (Kir) channel subunits Kir2.1, Kir4.1 and Kir7.1 in native bovine RPE and neural retina. The distribution of Kir2.1, Kir4.1 and Kir7.1 protein was determined in frozen sections of bovine, monkey, human and developing murine retinae by immunofluorescence and ABC immunohistochemistry.Results. RT-PCR analysis revealed Kir7.1 transcript in both RPE and neural retina, but Kir2.1and Kir4.1 transcript in the neural retina alone. In Northern blots, Kir2.1 and Kir4.1 probe hybridized to an appropriately sized-transcript in neural retina but not in RPE. Kir7.1 probe hybridized to a major transcript of~1.5 Kb in both RPE and neural retina, but with greater expression in RPE. In Western blots, Kir7.1 antibody recognized a major monomer of ~53KDa in RPE, while Kir2.1and Kir4.1 antibody recognized a monomer of~60 KDa in neural retina but not in RPE. Immunofluorescence revealed Kir2.1 and Kir4.1 immullostaining was expressed at many parts of muller cells, as Kir2.1especially in the membrane domains of muller cells that contact retinal neurons, ie, along the two stem processes over the soma, and in the side branches extending into the synaptil layers. No immunostaining was seen in RPE. Double staining that Kir2.1 and glutamine synthetase, Kir2.1 and glutamine synthetase colocalized well. Intense Kir7.1 immunolabeling was present on the apical surface of all RPE cells and appeared to extend over the length of the apical processes. Na+, K+ -ATPase expression varied among RPE cells, but in highly expressing cells, it co-localized with Kir7.1. Immunoreactivity of Kir2.1?Kir4.1 and Kir7.1 acomplished by ABC immunohistochemistry in monkey and human retinae got the nearly same results as bovine . Expression of Kir2.1 and Kir4.1 channel subunits in retinae got nearly same pattern as adults at P16 murine retinae, for Kir7.1, nearly same property as adults at P7 murine RPE.Conclusions. These results indicate that Kir2.1 and Kir4.1 channel subunits, nearly same property as adults at P16 murine retinae, is in muller cells , not in RPE. There is no difference between species. The Kir7.1 channel subunit, nearly same property as adults at P7 murine RPE, is a major component of the apical K+ conductance in bovine RPE. Kir7.1 is distributed over the length of apical processes, where it likely functions in the regulation of K+ transport and the electrical response of the RPE to light-evoked changes in subretinal K+ concentration.