發(fā)布時(shí)間：2018-08-28 17:51

【摘要】：背景：耳聾是人類(lèi)發(fā)病率最高的感官或功能缺陷性疾病,極大比例的耳聾與遺傳有關(guān)。目前仍有眾多耳聾基因未被鑒定,因此發(fā)現(xiàn)新的耳聾基因、揭示其致聾機(jī)制是聽(tīng)覺(jué)功能障礙研究的熱點(diǎn)。精子相關(guān)抗原6(sperm associated antigen 6,spag6)基因編碼纖毛相關(guān)蛋白SPAG6,最早發(fā)現(xiàn)于罹患不育癥的男性患者體內(nèi)。研究發(fā)現(xiàn),Spag6基因敲除小鼠精子鞭毛中心微管喪失,該小鼠出現(xiàn)精子運(yùn)動(dòng)障礙及不育。此外,SPAG6尚且存在于其他含有纖毛細(xì)胞的組織和器官中,比如中樞神經(jīng)系統(tǒng)、呼吸系統(tǒng)、血液系統(tǒng)甚至部分腫瘤。Spag6基因的異�？梢詫�(dǎo)致諸如腦積水、肺炎、生長(zhǎng)發(fā)育遲緩等多種疾病。這表明Spag6基因?qū)τ诨铙w內(nèi)多種重要生理功能的維持起著至關(guān)重要的作用。眾所周知柯蒂氏器(organ of Corti's)中的毛細(xì)胞對(duì)聽(tīng)覺(jué)的產(chǎn)生極其重要。其中圓柱形的外毛細(xì)胞在接受聲音的機(jī)械刺激后能迅速改變其自身的長(zhǎng)短和韌度,這源于一種被稱作prestin的動(dòng)力性蛋白分子。Prestin是一種高度特化的陰離子載體,屬于溶質(zhì)轉(zhuǎn)運(yùn)體超家族SLC26 (solute carrier protein),并特異地分布于內(nèi)耳柯蒂氏器外毛細(xì)胞(outer hair cells, OHCs)的側(cè)壁,它是外毛細(xì)胞電致運(yùn)動(dòng)和聽(tīng)覺(jué)放大效應(yīng)的分子基礎(chǔ),有馬達(dá)蛋白的別稱。Prestin的表達(dá)受到多因素的影響,如使用耳毒性藥物、遭受噪音刺激、接受放射線照射、與某些蛋白的直接相互作用等。因此,依賴于prestin的正常聽(tīng)覺(jué)對(duì)多種因素的干擾十分敏感。原發(fā)性纖毛運(yùn)動(dòng)障礙患者往往同時(shí)存在聽(tīng)覺(jué)障礙,提示編碼纖毛的某些蛋白可能在聽(tīng)覺(jué)生理過(guò)程中起到了一定作用。SPAG6廣泛表達(dá)于纖毛細(xì)胞中并且與內(nèi)耳發(fā)育密切相關(guān),故據(jù)此推測(cè)這種微管相關(guān)蛋白可能表達(dá)于內(nèi)耳毛細(xì)胞中而且對(duì)于毛細(xì)胞的功能至關(guān)重要。我們?cè)谇捌谘芯恐邪l(fā)現(xiàn),與野生型(Spag6+/+)和雜合型(Spag6+/-)小鼠相比,Spag基因敲除(Spag6-/-)小鼠出現(xiàn)重度耳聾。因此,SPAG6如果在OHCs中表達(dá),很可能參與電致運(yùn)動(dòng)并和prestin相關(guān)聯(lián)。目前關(guān)于prestin和微管蛋白相關(guān)性的研究鮮見(jiàn)報(bào)導(dǎo)。故本研究利用Spag基因敲除小鼠,探索SPAG6在毛細(xì)胞中的表達(dá)以及prestin與SPAG6之間的可能聯(lián)系。目的：探究SPAG6是否在內(nèi)耳毛細(xì)胞中表達(dá),如果有表達(dá),研究SPAG6和prestin之間的可能聯(lián)系。材料和方法：以雌、雄雜合型小鼠(Spag6+/-)交配并繁殖得到子代野生型(Spag6+/+)、雜合型(Spag6+/-)和基因敲除(Spag6-/-)小鼠,并根據(jù)不同基因型進(jìn)行分組研究。利用免疫熒光染色技術(shù),在共聚焦顯微鏡下觀察柯蒂氏器毛細(xì)胞的形態(tài)特點(diǎn)以及SPAG6在毛細(xì)胞中的分布情況。通過(guò)蛋白印跡實(shí)驗(yàn)和實(shí)時(shí)定量聚合酶鏈?zhǔn)椒磻?yīng)來(lái)測(cè)定prestin在不同基因型小鼠(Spag6+/+,Spag6+/-,Spag6-/-)中的表達(dá)量。以免疫共沉淀實(shí)驗(yàn)驗(yàn)證prestin和SPAG6兩種蛋白之間的可能聯(lián)系。結(jié)果：1.SPAG6表達(dá)于外毛細(xì)胞的表皮板內(nèi),同時(shí)SPAG6與prestin-起表達(dá)于外毛細(xì)胞的外側(cè)壁處。2.與野生型和雜合型小鼠相比,基因敲除小鼠prestin的熒光強(qiáng)度低,并且其外毛細(xì)胞有明顯的形態(tài)異常。3.基因敲除小鼠(Spag6-/-)prestin的mRNA以及蛋白表達(dá)量均顯著降低。4.免疫共沉淀實(shí)驗(yàn)顯示prestin和SPAG6之間存在相互作用。結(jié)論：成功繁殖出了Spag6基因突變小鼠,其體貌和基因特征與先前研究一致,為后續(xù)實(shí)驗(yàn)做好了準(zhǔn)備。通過(guò)維持prestin的正常表達(dá),SPAG6對(duì)外毛細(xì)胞結(jié)構(gòu)的穩(wěn)定些起到了至關(guān)重要的作用�；蚯贸托∈蟪霈F(xiàn)重度耳聾,表明Spag6基因與耳聾密切相關(guān)。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R764.43

【共引文獻(xiàn)】

相關(guān)期刊論文 前4條

1 韓明昱;戴樸;;我國(guó)耳聾基因診斷的臨床應(yīng)用進(jìn)展[J];北京醫(yī)學(xué);2011年05期

2 余紅;楊晶群;劉丹;吳志強(qiáng);;聽(tīng)力初篩未通過(guò)新生兒常見(jiàn)聾病易感基因檢測(cè)結(jié)果分析[J];中國(guó)兒童保健雜志;2014年10期

3 羅紅強(qiáng);黃妍雯;李文龍;劉軍;;南昌地區(qū)耳聾患者耳聾基因診斷及早期干預(yù)[J];實(shí)用臨床醫(yī)學(xué);2012年01期

4 杜娟;許涓涓;黃萍麗;付華鈺;;南寧市4679例新生兒突變耳聾基因篩查結(jié)果[J];山東醫(yī)藥;2015年43期

相關(guān)博士學(xué)位論文 前4條

1 韓明昱;基于基因診斷的遺傳性耳聾預(yù)防與干預(yù)研究[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2011年

2 陳俞;新疆維吾爾族和漢族非綜合征性聾耳聾基因常見(jiàn)突變的研究[D];新疆醫(yī)科大學(xué);2011年

3 王國(guó)建;耳聾基因診斷的臨床實(shí)踐[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2008年

4 盧彥平;幾種染色體病及單基因病的產(chǎn)前遺傳學(xué)診斷技術(shù)研究[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2010年

相關(guān)碩士學(xué)位論文 前4條

1 席宏;大前庭水管綜合征SLC26A4基因突變臨床研究[D];山西醫(yī)科大學(xué);2011年

2 張強(qiáng)偉;129例非綜合征型耳聾患者耳聾基因的篩查[D];山西醫(yī)科大學(xué);2012年

3 胡煜;非綜合征型聾患者GJB2基因、GJB3基因、SLC26A4基因、線粒體12S rRNA基因突變分析[D];安徽醫(yī)科大學(xué);2012年

4 劉啟珍;重慶市永川地區(qū)青少年耳聾患者耳聾基因熱點(diǎn)突變篩查分析[D];重慶醫(yī)科大學(xué);2013年

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本文編號(hào)：2210124