ARL2在视网膜中的功能研究

发布时间：2018-11-02 06:50

【摘要】：视网膜变性(retinal degeneration)是由感光细胞异常引起的一类疾病的统称,是致盲疾病的重要因素之一。在人类视网膜变性疾病中,发现的突变基因有很大一部分由于影响了蛋白在感光细胞中的运输而导致感光细胞死亡的。目前尚无有效的预防和治疗措施,发现新的致病基因和致病机理可为临床诊疗提供重要依据。Arl2基因是Arl基因家族的一个成员,它编码20kDa的GTP水解酶,在真核生物中高度保守且普遍表达,参与微管蛋白折叠、与线粒体的运动性相关。感光细胞内异戊二烯化蛋白运输到感光细胞外节段需要PDEδ的参与及其变构调节因子ARL2的调控释放。本课题旨在对Arl2条件性敲除小鼠进行表型的初步鉴定,研究ARL2是否参与感光细胞内异戊二烯化蛋白等膜蛋白的运输,以及在视网膜中的对线粒体、微管蛋白的影响。通过利用Cre/Loxp和Frt/Flp重组系统,获得特异Arl2基因敲除的小鼠。采用视网膜电图方法检测、免疫组织化学方法、激光共聚焦扫描显微镜观察,透射电镜拍摄等技术鉴定小鼠的初步表型,视网膜中膜蛋白(如PDE6D、GRK1、GC1、MLopsin等)的表达量以及在细胞内的定位是否发生改变,并判定视网膜的组织形态学和功能是否受影响。与野生型小鼠相比,Arl2-/-小鼠在P12天时体型和眼睛都明显较小。在P30,Arl2基因敲除小鼠的感光细胞死亡,内节、外节缩短。视网膜变薄,细胞层数因凋亡而明显减少,并检测到凋亡信号。敲除小鼠在明适应和暗适应中视网膜电生理反应性均降低,暗示了视锥视杆细胞数量减少或功能损坏。TM(rhodopsin、opsin、GC1、CNGA1/3)和PM(Tα、GRK1、PDE6)蛋白的表达水平略微下调,其中Rhodopsin和INPP5E蛋白的荧光染色观察到少量异常定位,多数蛋白但并未出现运输异常。另外,Arl2基因敲除还导致线粒体和微管蛋白表达水平下调;并意外地发现敲除小鼠不能形成完整的血管结构。根据以上得出结论,ARL2对维持感光细胞存活和视网膜结构功能的完整性至关重要,并对感光细胞的外节的正常发育起重要作用;ARL2对大多数膜蛋白的运输不是必需的,但对Rhodopsin、INPP5E的胞内运输起微调作用——ARL2可能是Rhodopsin、INPP5E的调节释放因子;ARL2还在视网膜血管发育中发挥重要作用,具体机制还有待进一步研究;Arl2基因在眼睛中的敲除通过凋亡,使微管解聚、线粒体减少,对膜蛋白的运输异常三个途径导致视网膜变性。
[Abstract]:Retinal degeneration (retinal degeneration) is a kind of disease caused by abnormal photoreceptor cells, which is one of the important factors leading to blindness. In human retinal degeneration, a large part of the mutated genes are found to cause the death of photoreceptor cells due to their influence on the transport of proteins in photosensitive cells. At present, there are no effective prevention and treatment measures. The discovery of new pathogenic genes and pathogenesis may provide important basis for clinical diagnosis and treatment. Arl2 gene is a member of Arl gene family, which encodes GTP hydrolase of 20kDa. It is highly conserved and widely expressed in eukaryotes and is involved in the folding of tubulin, which is related to the motility of mitochondria. The transport of isoprene protein from photosensitive cells to the outer segment of photosensitive cells requires the participation of PDE 未 and the regulation and release of ARL2. The aim of this study was to identify the phenotypes of Arl2 conditioned knockout mice and to investigate whether ARL2 is involved in the transport of isoprene and other membrane proteins in photoreceptor cells and the effects of ARL2 on mitochondria and tubulin in the retina. The specific Arl2 gene knockout mice were obtained by using Cre/Loxp and Frt/Flp recombination systems. The primary phenotype and retinal membrane proteins (such as PDE6D,GRK1,GC1,) of mice were identified by electroretinogram, immunohistochemistry, laser confocal scanning microscopy and transmission electron microscopy. Whether the expression and localization of MLopsin were changed, and whether the histomorphology and function of retina were affected. Compared with wild-type mice, Arl2-/- mice were significantly smaller in size and eyes at P 12 d. The photosensitive cells of P30 + Arl2 knockout mice died, and the internal and outer segments were shortened. As the retina thinned, the number of cell layers was significantly reduced and apoptotic signals were detected. The retina electrophysiological reactivity of knockout mice decreased in both light and dark adaptation, suggesting that the number of conical rod cells decreased or the function damaged. TM (rhodopsin,opsin,GC1,CNGA1/3) and PM (T 伪, GRK1,. The expression level of PDE6) protein was slightly down-regulated. A small amount of abnormal localization was observed by fluorescence staining of Rhodopsin and INPP5E proteins, but no abnormal transport was found in most of the proteins. In addition, Arl2 knockout also resulted in the down-regulation of mitochondrial and tubulin expression, and accidentally found that knockout mice could not form a complete vascular structure. According to the above conclusions, ARL2 plays an important role in maintaining the survival of photoreceptor cells and the integrity of retinal structure and function, and plays an important role in the normal development of the outer ganglia of photoreceptor cells. ARL2 is not necessary for the transport of most membrane proteins, but it has a fine-tuning effect on the intracellular transport of Rhodopsin,INPP5E-ARL2 may be the regulatory releasing factor of Rhodopsin,INPP5E. ARL2 also plays an important role in retinal vascular development, and the specific mechanism remains to be further studied. The knockout of Arl2 gene in the eyes leads to retinal degeneration through apoptosis, depolymerization of microtubules, decrease of mitochondria and abnormal transport of membrane proteins.
【学位授予单位】：电子科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R774.1

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