关于Dlicl基因在小鼠视网膜色素变性中的功能研究

发布时间：2018-04-27 10:13

本文选题：动力蛋白 + Dlic1　；参考：《复旦大学》2013年博士论文

【摘要】：视网膜色素变性(retinitis pigmentosa)是一种遗传性的致盲疾病,是山于视网膜感光细胞(photoreceptor cell)死亡而导致的视力受损。目前已经确定了50多个致病基因的突变位点,但尚无有效的预防和治疗措施。发现新的致病基因并研究其在视网膜色素变性中的作用和机制将为临床治疗提供重要的理论基础。动力蛋白(dynein)是向微管负端运动的一类马达蛋白,在细胞内对于蛋白质运输、细胞器定位具有重要的作用。Dlicl基因编码哺乳动物动力蛋白轻链中间链1,是动力蛋白中的一个亚基。通过建立Dlicl基因敲除小鼠,我们研究了该基因在小鼠中的功能。我们发现Dlic-/-小鼠可以存活,但该小鼠的感光细胞内外节发育不完全并且逐渐发生凋亡。利用该基因敲除小鼠我们进一步研究了感光细胞发生凋亡的机制以及Dlicl在感光细胞中的功能。免疫荧光检测发现,在Dlicl感光细胞内合成的外节蛋白质异位累积在胞体内。蛋白质从内质网到高尔基的运输受阻碍,导致内质网网腔膨大,但是膨大的内质网网腔并末激活内质网应答反应。Rabll囊泡介导蛋白质从高尔基体运输到纤毛,Dlicl缺失导致Rab11囊泡积累在细胞核周围的胞体中。这说明Dlicl感光细胞中蛋白质从高尔基体到纤毛的运输受阻。进一步的研究表明,Dlicl基因缺失导致动力蛋白重链和中间链蛋白水平降低,并且在感光细胞内节的含量减少,暗示了胞内动力蛋白的运输能力降低。同时,动力蛋白结合Rabll囊泡的能力被减弱,因此而导致Rab11介导的囊泡不能有效运输而发生累积。另外,Dlicl基因缺失影响了感光细胞纤毛发育。而纤毛是蛋白质从内节到外节运输的重要通道,运输通道的异常发育进一步加剧了外节蛋白在胞体内的累积。根据以上的新发现我们得出结论,Dlicl敲除造成感光细胞动力蛋白减少和异常分布,一方面降低了动力蛋白运载囊泡的能力,使得高尔基到纤毛的囊泡运输发生阻碍。另一方面影响了感光细胞纤毛发育异常。这两方面共同作用,使得细胞合成的外节蛋白异位累积在胞体内,造成外节发育不全并且逐渐发生凋亡。因此,Dlicl-/-小鼠为研究人类视网膜色素变性的机理提供了新的动物模型。
[Abstract]:Retinitis pigmentosais a hereditary and blinding disease, which is caused by the death of photoreceptor cells in the retina. More than 50 mutation sites have been identified, but there are no effective prevention and treatment measures. The discovery of new pathogenic genes and the study of their roles and mechanisms in retinitis pigmentosa will provide an important theoretical basis for clinical treatment. Dynein) is a kind of motor protein which moves to the negative end of microtubule. It plays an important role in intracellular protein transport and organelle localization. Dlicl gene encodes light chain intermediate chain 1 of mammalian dynamic protein, which is a subunit of dynamic protein. By establishing Dlicl knockout mice, we studied the function of Dlicl gene in mice. We found that Dlic-r-mice could survive, but the photosensitive cells of the mice were not fully developed and gradually apoptotic. Using this gene knockout mouse we further studied the mechanism of photoreceptor cell apoptosis and the function of Dlicl in photoreceptor cells. Immunofluorescence assay showed that the ectopic proteins synthesized in Dlicl photosensitive cells accumulated in vivo. The transport of proteins from the endoplasmic reticulum to Golgi is hindered, leading to enlargement of the endoplasmic reticulum cavity. But the enlarged endoplasmic reticulum cavity does not activate the endoplasmic reticulum response. Rabll vesicle mediates the transport of proteins from Golgi body to cilia, resulting in the accumulation of Rab11 vesicles in the perinuclear cell bodies. This suggests that the transport of proteins from Golgi body to cilium in Dlicl photosensitive cells is blocked. Further studies showed that the deletion of Dlicl gene resulted in a decrease in the levels of heavy chain and intermediate chain proteins, and a decrease in the content of intracellular knots in photoreceptor cells, suggesting a decrease in the transport capacity of intracellular dynamic proteins. At the same time, the ability of dynamic protein binding to Rabll vesicles was weakened, which led to the accumulation of Rab11 mediated vesicles that could not transport effectively. In addition, the deletion of Dlicl gene affected the cilia development of photoreceptor cells. Cilium is an important channel for the transport of proteins from internal to outer nodes, and the abnormal development of transport channels further intensifies the accumulation of outer node proteins in the body. Based on the above findings, we conclude that Dlicl knockout results in the decrease and abnormal distribution of photoreceptor cell motility proteins, which on the one hand reduces the ability of motility proteins to carry vesicles, and impedes the transport of Golgi to ciliated vesicles. On the other hand, it affects the abnormal development of the cilia of photosensitive cells. These two factors make the ectopic accumulation of extracellular nodal proteins in the cell, resulting in hypoplasia of the outer node and gradual apoptosis. Therefore, Dlicl-r-mice provide a new animal model for studying the mechanism of human retinitis pigmentosa.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774.13

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