Prestin在Spag6基因敲除小鼠外毛细胞中的表达研究

发布时间：2018-08-28 17:51

【摘要】：背景：耳聋是人类发病率最高的感官或功能缺陷性疾病,极大比例的耳聋与遗传有关。目前仍有众多耳聋基因未被鉴定,因此发现新的耳聋基因、揭示其致聋机制是听觉功能障碍研究的热点。精子相关抗原6(sperm associated antigen 6,spag6)基因编码纤毛相关蛋白SPAG6,最早发现于罹患不育症的男性患者体内。研究发现,Spag6基因敲除小鼠精子鞭毛中心微管丧失,该小鼠出现精子运动障碍及不育。此外,SPAG6尚且存在于其他含有纤毛细胞的组织和器官中,比如中枢神经系统、呼吸系统、血液系统甚至部分肿瘤。Spag6基因的异常可以导致诸如脑积水、肺炎、生长发育迟缓等多种疾病。这表明Spag6基因对于活体内多种重要生理功能的维持起着至关重要的作用。众所周知柯蒂氏器(organ of Corti's)中的毛细胞对听觉的产生极其重要。其中圆柱形的外毛细胞在接受声音的机械刺激后能迅速改变其自身的长短和韧度,这源于一种被称作prestin的动力性蛋白分子。Prestin是一种高度特化的阴离子载体,属于溶质转运体超家族SLC26 (solute carrier protein),并特异地分布于内耳柯蒂氏器外毛细胞(outer hair cells, OHCs)的侧壁,它是外毛细胞电致运动和听觉放大效应的分子基础,有马达蛋白的别称。Prestin的表达受到多因素的影响,如使用耳毒性药物、遭受噪音刺激、接受放射线照射、与某些蛋白的直接相互作用等。因此,依赖于prestin的正常听觉对多种因素的干扰十分敏感。原发性纤毛运动障碍患者往往同时存在听觉障碍,提示编码纤毛的某些蛋白可能在听觉生理过程中起到了一定作用。SPAG6广泛表达于纤毛细胞中并且与内耳发育密切相关,故据此推测这种微管相关蛋白可能表达于内耳毛细胞中而且对于毛细胞的功能至关重要。我们在前期研究中发现,与野生型(Spag6+/+)和杂合型(Spag6+/-)小鼠相比,Spag基因敲除(Spag6-/-)小鼠出现重度耳聋。因此,SPAG6如果在OHCs中表达,很可能参与电致运动并和prestin相关联。目前关于prestin和微管蛋白相关性的研究鲜见报导。故本研究利用Spag基因敲除小鼠,探索SPAG6在毛细胞中的表达以及prestin与SPAG6之间的可能联系。目的：探究SPAG6是否在内耳毛细胞中表达,如果有表达,研究SPAG6和prestin之间的可能联系。材料和方法：以雌、雄杂合型小鼠(Spag6+/-)交配并繁殖得到子代野生型(Spag6+/+)、杂合型(Spag6+/-)和基因敲除(Spag6-/-)小鼠,并根据不同基因型进行分组研究。利用免疫荧光染色技术,在共聚焦显微镜下观察柯蒂氏器毛细胞的形态特点以及SPAG6在毛细胞中的分布情况。通过蛋白印迹实验和实时定量聚合酶链式反应来测定prestin在不同基因型小鼠(Spag6+/+,Spag6+/-,Spag6-/-)中的表达量。以免疫共沉淀实验验证prestin和SPAG6两种蛋白之间的可能联系。结果：1.SPAG6表达于外毛细胞的表皮板内,同时SPAG6与prestin-起表达于外毛细胞的外侧壁处。2.与野生型和杂合型小鼠相比,基因敲除小鼠prestin的荧光强度低,并且其外毛细胞有明显的形态异常。3.基因敲除小鼠(Spag6-/-)prestin的mRNA以及蛋白表达量均显著降低。4.免疫共沉淀实验显示prestin和SPAG6之间存在相互作用。结论：成功繁殖出了Spag6基因突变小鼠,其体貌和基因特征与先前研究一致,为后续实验做好了准备。通过维持prestin的正常表达,SPAG6对外毛细胞结构的稳定些起到了至关重要的作用。基因敲除型小鼠出现重度耳聋,表明Spag6基因与耳聋密切相关。
[Abstract]:BACKGROUND: Deafness is one of the most common sensory or functional disorders in humans, and a large proportion of deafness is related to heredity. There are still many deafness genes that have not been identified, so finding new deafness genes and revealing the mechanism of deafness are hot topics in the study of hearing impairment. SPAG6 was first found in infertile men because it encodes the cilia-associated protein SPAG6. Studies have shown that the sperm of Spag6 knockout mice loses microtubules in the flagellum center, and the mice develop sperm dyskinesia and infertility. In addition, SPAG6 also exists in other tissues and organs containing cilia, such as the central nervous system, respiration. Abnormalities in the Spag6 gene can lead to diseases such as hydrocephalus, pneumonia, and growth retardation. This suggests that the Spag6 gene plays an important role in maintaining many important physiological functions in vivo. Hair cells in the organ of Corti's are known to produce hearing. The cylindrical outer hair cells, which undergo mechanical stimulation of sound, rapidly change their length and toughness due to a dynamic protein molecule called prestin, a highly specialized anion carrier belonging to the solute transporter superfamily SLC26 (solute carrier protein), and specifically The lateral wall of the outer hair cells (OHCs) of the inner ear, which is the molecular basis for the electrokinesis and auditory amplification of the outer hair cells, has a motor protein nickname. Prestin expression is influenced by many factors, such as the use of ototoxic drugs, noise stimulation, radiation exposure, and direct interaction with certain proteins. Therefore, prestin-dependent normal hearing is very sensitive to interference from a variety of factors. Primary ciliary dyskinesia patients are often accompanied by hearing impairment, suggesting that some of the proteins encoding cilia may play a role in auditory physiology. SPAG6 is widely expressed in cilia cells and closely related to inner ear development. Therefore, we speculate that this microtubule-associated protein may be expressed in inner ear hair cells and is essential for hair cell function. In our previous study, we found that compared with wild type (Spag6+/+) and heterozygous type (Spag6+/-) mice, Spag gene knockout (Spag6-/-) mice developed severe deafness. There are few reports about the relationship between prestin and tubulin. So this study used Spag knockout mice to explore the expression of SPAG6 in hair cells and the possible relationship between prestin and SPAG6. Materials and Methods: Female and male heterozygous mice (Spag6+/-) were used to mate and reproduce wild type (Spag6+/+), heterozygous type (Spag6+/-) and gene knockout (Spag6-/-) mice, and were grouped according to different genotypes. Morphological characteristics of Cotty's hair cells and the distribution of SPAG6 in hair cells. The expression of prestin in different genotypes of mice (Spag6+/+, Spag6+/-, Spag6-/-) was determined by Western blotting and real-time quantitative polymerase chain reaction. The possible association between prestin and SPAG6 was verified by immunoprecipitation assay. Results: 1. SPAG6 was expressed in the epidermis of outer hair cells, and SPAG6 and prestin-began to express in the lateral wall of outer hair cells. 2. Compared with wild-type and heterozygous mice, the fluorescence intensity of prestin in gene knockout mice was lower, and the morphological abnormalities of outer hair cells were obvious. 3. The expression of SPAG6-prestin mRNA and the expression of prestin in gene knockout mice (Spag6-/-) The expression of SPAG6 protein was significantly decreased. 4. Immunocoprecipitation test showed that there was interaction between SPAG6 and prestin. Conclusion: Spag6 mutant mice were successfully bred, and their physiognomy and genetic characteristics were consistent with previous studies, which prepared for further experiments. These play a crucial role. Severe deafness in knockout mice suggests that the Spag6 gene is closely related to deafness.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R764.43

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