Müller细胞钾离子通道Kir4.1在视网膜变性疾病中的作用及其调控机制的研究

Retinitis pigmentosa(RP) is a severe sight-threanting disease.The accumulation of extracellular glutamate has been demonstrated as an important cause of progression of RP. It has been showed that transportation of extracellular glutamate was closely associated with function of Kir4.1 channel of astrocytes in central nervous system (CNS). Our previous study in RCS rats (RP animal model) showed that the expression and function of specific potassium channel-Kir4.1 in Müller cells increased transiently, then dropped down. While the effect and regulation mechanism of Kir4.1 in RP is still unknown. Our hypothesis is: metabotropic glutamate receptors (mGluR1) of Müller cells is activated in RP, followed by down-regulation of Kir4.1 through GPCRs, then results in accumulation of extracellular glutamate and aggravates retinal degeneration. We propose to perform subretinal injection of Kir4.1-shRNA or guanosine, intravitreal injection of agonist or antagonist of mGluR1, then evaluate expression of Kir4.1 and GLAST, extracellular glutamate concentration, morphology and function of retinal neurons. In co-culture of Müller cells and photoreptors, we observe activity of photoreptors after RNAi of Kir4.1 or treatment with guanosine. Success in this project would facilitate our deeper understanding of effect and regulation mechanism of Kir4.1 in retinal degeneration, and introduce novel therapeutic approaches for the treatment of RP.

视网膜色素变性是严重致盲眼病，胞外微环境中谷氨酸聚积是病变进展重要因素。CNS中胞外谷氨酸转运与星型胶质细胞Kir4.1通道的作用密切相关。我们前期研究发现：视网膜变性大鼠中，Müller细胞Kir4.1表达及功能先增强后降低，但其在视网膜变性中的作用及调控不明。由此假设：视网膜变性中Müller细胞代谢型谷氨酸受体mGluR1激活后，经GPCRs下调Kir4.1活性，使胞外谷氨酸堆积，加重视网膜变性。拟研究大鼠视网膜下注射Kir4.1-shRNA或鸟苷以及玻璃体腔注射mGluR1激动剂或拮抗剂后, Müller细胞Kir4.1、GLAST和胞外谷氨酸浓度，视网膜神经元形态和功能；并采用Müller细胞和光感受器细胞共培养，用RNAi或加入鸟苷以及加入mGluR1激活或拮抗剂后，观察光感受器活性。探讨Müller细胞Kir4.1在视网膜变性中的作用及调控机制,为寻找可能的治疗靶点提供依据。

视网膜色素变性是严重致盲眼病,胞外微环境中谷氨酸聚积是病变进展重要因素。我们发现RCS大鼠变性过程中Müller细胞的GS、Kir4.1表达降低，而谷氨酸的浓度则升高，mGluR5的表达也明显升高。我们接下来进一步探讨了mGluR5、GS、Kir4.1三者之间的关系。在RCS鼠的对照大鼠视网膜下腔注射DL-AAA，一种特异性阻断GS的药物，发现ERG的a、b波幅值均降低，但其比值并未变化，说明b波的降低是源于a波。也就是说，特异性的阻断GS，会导致视网膜外层的光反应能力降低。我们实验发现抑制GS后，mGluR5而不是mGluR1表达升高。选择mGluR5的特异性激动剂和拮抗剂，同样通过视网膜下腔注射发现，激动mGluR5导致a波幅值降低，而抑制mGluR5则会引起a波升高。实验证明mGluR5的下游通路是传统的Gαq-Ca2+通路。进一步的研究发现，mGluR5可以调控K电流的幅值，同时影响Kir4.1和AQP4的表达，三者形成了一个大分子复合物。最后在RCS大鼠中，特异性的抑制mGluR5可以部分改善视网膜的功能，采用多次给药的方式可以延长该效用，提示mGluR5可能是治疗视网膜变性治疗的一个药物靶点。该项目主要研究结果共发表文章4篇，其中国内核心期刊3篇、SCI论著1篇。参加会议3次，作分会场口头报告。该项目培养博士研究生1名，硕士研究生1名。