【摘要】：視網膜變性(retinal degeneration)是由感光細胞異常引起的一類疾病的統(tǒng)稱,是致盲疾病的重要因素之一。在人類視網膜變性疾病中,發(fā)現(xiàn)的突變基因有很大一部分由于影響了蛋白在感光細胞中的運輸而導致感光細胞死亡的。目前尚無有效的預防和治療措施,發(fā)現(xiàn)新的致病基因和致病機理可為臨床診療提供重要依據(jù)。Arl2基因是Arl基因家族的一個成員,它編碼20kDa的GTP水解酶,在真核生物中高度保守且普遍表達,參與微管蛋白折疊、與線粒體的運動性相關。感光細胞內異戊二烯化蛋白運輸?shù)礁泄饧毎夤?jié)段需要PDEδ的參與及其變構調節(jié)因子ARL2的調控釋放。本課題旨在對Arl2條件性敲除小鼠進行表型的初步鑒定,研究ARL2是否參與感光細胞內異戊二烯化蛋白等膜蛋白的運輸,以及在視網膜中的對線粒體、微管蛋白的影響。通過利用Cre/Loxp和Frt/Flp重組系統(tǒng),獲得特異Arl2基因敲除的小鼠。采用視網膜電圖方法檢測、免疫組織化學方法、激光共聚焦掃描顯微鏡觀察,透射電鏡拍攝等技術鑒定小鼠的初步表型,視網膜中膜蛋白(如PDE6D、GRK1、GC1、MLopsin等)的表達量以及在細胞內的定位是否發(fā)生改變,并判定視網膜的組織形態(tài)學和功能是否受影響。與野生型小鼠相比,Arl2-/-小鼠在P12天時體型和眼睛都明顯較小。在P30,Arl2基因敲除小鼠的感光細胞死亡,內節(jié)、外節(jié)縮短。視網膜變薄,細胞層數(shù)因凋亡而明顯減少,并檢測到凋亡信號。敲除小鼠在明適應和暗適應中視網膜電生理反應性均降低,暗示了視錐視桿細胞數(shù)量減少或功能損壞。TM(rhodopsin、opsin、GC1、CNGA1/3)和PM(Tα、GRK1、PDE6)蛋白的表達水平略微下調,其中Rhodopsin和INPP5E蛋白的熒光染色觀察到少量異常定位,多數(shù)蛋白但并未出現(xiàn)運輸異常。另外,Arl2基因敲除還導致線粒體和微管蛋白表達水平下調;并意外地發(fā)現(xiàn)敲除小鼠不能形成完整的血管結構。根據(jù)以上得出結論,ARL2對維持感光細胞存活和視網膜結構功能的完整性至關重要,并對感光細胞的外節(jié)的正常發(fā)育起重要作用;ARL2對大多數(shù)膜蛋白的運輸不是必需的,但對Rhodopsin、INPP5E的胞內運輸起微調作用——ARL2可能是Rhodopsin、INPP5E的調節(jié)釋放因子;ARL2還在視網膜血管發(fā)育中發(fā)揮重要作用,具體機制還有待進一步研究;Arl2基因在眼睛中的敲除通過凋亡,使微管解聚、線粒體減少,對膜蛋白的運輸異常三個途徑導致視網膜變性。
[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 偽, 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.
【學位授予單位】：電子科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R774.1

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