RNF8基因敲除对小鼠耳蜗老化机制影响的研究

发布时间：2018-03-30 14:34

本文选题：RNF8　切入点：DNA损伤修复　出处：《兰州大学》2017年硕士论文

【摘要】：目的:RNF8是一种泛素连接酶E3,在DNA损伤修复中发挥重要作用,参与细胞凋亡、衰老、突变反应和神经退行性变,肿瘤的发生。本研究通过观察不同月龄RNF8基因敲除型(KO)与野生型(WT)小鼠之间耳蜗毛细胞、血管纹、螺旋神经节细胞形态、数量、密度、厚度、损伤定位等变化,探讨RNF8在DNA损伤修复中的作用及RNF8缺失后小鼠耳蜗主要结构的老化机制,为老年性耳聋病理和治疗提供依据。另对小鼠耳蜗取材和包埋技术进行了改良。方法:通过配对繁殖获取基因敲除小鼠和同窝野生对照。至8周龄、16周龄,32周龄分组。用小鼠耳蜗取材和包埋改良技术进行组织切片,HE、CV染色观察耳蜗毛细胞、血管纹、螺旋神经节细胞细胞密度、数量、厚度的变化;免疫荧光γ-H2AX染色;免疫组化8-oHdG标记DNA损伤的程度及表达量;脂褐素、β-半乳糖苷酶染色检测受损细胞衰老。结果:1.小鼠耳蜗改良取材技术实验结果时间提高72.4%,完好率提高41.4%。切片完好率37.2%。2.HE、CV染色发现,同龄KO组比WT组毛细胞改变明显。血管纹三层出现细胞数量减少,空泡,分离、断裂。螺旋神经节细胞缺失明显,严重裂隙。3.耳蜗增龄性变化发现,RNF8 KO小鼠耳蜗毛细胞、血管纹、螺旋神经节增龄性改变更加明显,免疫荧光γ-H2AX染色显示损伤的细胞H2AX磷酸化反应。4.小鼠耳蜗老化改变,KO组β-半乳糖苷酶染色发现血管纹随增龄性改变32周龄为著,脂褐素染色发现细胞核周围棕褐色淡染。(P0.05)结论:改良快速小鼠耳蜗取材和切片包埋技术方法简便易行,成本低廉,节约资源,值得推广。RNF8缺失是导致小鼠耳蜗形态学方面改变的重要原因之一,也间接影响着耳蜗生理功能,随着年龄增加而愈加明显。RNF8对小鼠耳蜗各主要结构老化影响的差异化与老年性耳聋临床各表现型存在着密切关联,RNF8缺失加速小鼠耳蜗的老化。RNF8缺失对小鼠耳蜗组织的修复功能机制具有重要影响。RNF8基因缺陷小鼠耳蜗的凋亡和衰老密切相关。
[Abstract]:Objective: RNF8 is a ubiquitin ligase E3, which plays an important role in the repair of DNA damage and plays an important role in apoptosis, senescence, mutation reaction and neurodegenerative changes. In this study, the morphology, number, density, thickness and injury location of cochlear hair cells, stria vascularis and spiral ganglion cells were observed between RNF8 knockout type and wild type WT-mice at different ages. To explore the role of RNF8 in the repair of DNA damage and the aging mechanism of the main cochlear structures in mice after RNF8 deletion. In addition, the cochlea sampling and embedding techniques of mice were improved. Methods: gene knockout mice and the same nest wild control were obtained by paired propagation. The cochlea hair cells were observed by histopathological staining and HECV staining with the modified technique of mouse cochlea sampling and embedding. Changes of cell density, number and thickness of stria vascularis and spiral ganglion cells; immunofluorescence 纬 -H2AX staining; the degree and expression of DNA damage labeled with 8-oHdG immunohistochemical staining; Lipofuscin and 尾 -galactosidase staining were used to detect the aging of damaged cells. The changes of hair cells in the same age KO group were more obvious than those in WT group. The number of hair cells in the three layers of stria vascularis was decreased, vacuole, dissociation and rupture. The loss of spiral ganglion cells was obvious, and the serious fissure was 3.The cochlear hair cells of RNF8KO mice were found to be in the age change of cochlea. The age-related changes of stria vascularis and spiral ganglia were more obvious, and immunofluorescence 纬 -H2AX staining showed that the injured cells were phosphorylated by H2AX. 4. 尾 -galactosidase staining of mouse cochlea aging group showed that the stria vascularis changed with the age of 32 weeks. Lipofuscin staining showed that the method of rapid cochlear sampling and embedding was simple and easy, low cost and resource saving. The loss of .RNF8 is one of the important reasons for the morphological changes in mouse cochlea and indirectly affects the physiological function of the cochlea. The difference of aging effect of RNF8 on the main structure of mouse cochlea and clinical manifestations of presbycusis are closely related with age. RNF8 deletion accelerates cochlea aging in mice. RNF8 deletion affects cochlear group in mice. The mechanism of tissue repair has an important effect on cochlea apoptosis and senescence in mice with RNF8 gene deficiency.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R764.436

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