【摘要】：SLC26A4突變所致疾病為常染色體隱性遺傳,目前已發(fā)現(xiàn)200多種不同的突變類型,可導(dǎo)致Pendred綜合征和DFNB4(OMIM600791)非綜合征型遺傳性耳聾。Pendred綜合征臨床表現(xiàn)為甲狀腺腫大(甲狀腺功能正�；蜉p微降低)和耳聾。SLC26A4突變導(dǎo)致的耳聾常常伴隨有內(nèi)耳畸形,其中最常見的畸形是前庭導(dǎo)水管擴(kuò)大(enlarged vestibular aqueduct EVA)和Mondini畸形。Pendrin在內(nèi)耳主要參與Cl-、HCO3-的轉(zhuǎn)運(yùn),與維持耳蝸內(nèi)環(huán)境穩(wěn)定有關(guān)。另外,pendrin蛋白不直接調(diào)節(jié)鉀離子的跨膜轉(zhuǎn)運(yùn),但研究發(fā)現(xiàn)Pendrin對(duì)維持耳蝸內(nèi)電位(鉀離子平衡電位)產(chǎn)生聽覺起到至關(guān)重要的作用。本研究以本實(shí)驗(yàn)室首先發(fā)現(xiàn)的SLC26A4致耳聾突變S448X,以及構(gòu)建的S448X突變體和野生型SLC26A4與GFP融合基因表達(dá)質(zhì)粒為基礎(chǔ),分別體外轉(zhuǎn)染細(xì)胞,Western blot檢測(cè)驗(yàn)證蛋白表達(dá),然后直接觀察融合蛋白綠色熒光,同時(shí)對(duì)內(nèi)質(zhì)網(wǎng)(ER),高爾基體(Golgi),微管組織免疫熒光染色,利用免疫熒光的方法觀察突變體pendrin蛋白的亞細(xì)胞定位改變。另一方面,利用膜片鉗技術(shù)記錄分別表達(dá)野生型和突變體的細(xì)胞的全細(xì)胞電流的差異,明確SLC26A4基因突變對(duì)細(xì)胞氯離子和鉀離子轉(zhuǎn)運(yùn)能力的影響。 目的：觀察SLC26A4基因及其一個(gè)致聾突變S448X在體外細(xì)胞中的表達(dá)與功能改變,以探討其致病的可能機(jī)理。 方法：將已構(gòu)建完成的突變S448X和野生型SLC26A4與EGFP融合蛋白表達(dá)載體,轉(zhuǎn)染cos-7細(xì)胞,Western印跡分析蛋白的表達(dá),同時(shí)對(duì)內(nèi)質(zhì)網(wǎng),高爾基體,微管組織免疫熒光染色,激光共聚焦顯微鏡觀察突變蛋白和野生型SLC26A4的亞細(xì)胞定位改變。篩選穩(wěn)定表達(dá)突變S448X和野生型SLC26A4細(xì)胞,全細(xì)胞膜片鉗實(shí)驗(yàn)分析突變蛋白和野生型SLC26A4對(duì)細(xì)胞離子轉(zhuǎn)運(yùn)功能的影響。 結(jié)果：1、Western blot檢測(cè)SLC26A4野生型和S448X突變與EGFP融合蛋白在cOS-7細(xì)胞中的表達(dá)。真核表達(dá)質(zhì)粒pEGFP N1SLC26A4S448X和pEGFP N1SLC26A4WT能在cos-7細(xì)胞中進(jìn)行表達(dá),表達(dá)后的蛋白條帶大小與預(yù)期相符。S448X突變蛋白較野生型小,說明突變蛋白較野生型有截短。 2、pEGFP N1SLC26A4S448X在cos-7細(xì)胞中,綠色熒光主要分布在胞質(zhì)內(nèi)；并與內(nèi)質(zhì)網(wǎng)有共定位,與高爾基體、微管組織無共定位；細(xì)胞膜上無突變蛋白表達(dá)。pEGFP N1SLC26A4WT在cos-7細(xì)胞中野生型蛋白主要表達(dá)在細(xì)胞膜,可見部分綠色熒光聚集于細(xì)胞質(zhì)。 3、G418篩選得到的COS-7細(xì)胞系穩(wěn)定表達(dá)了野生型SLC26A4編碼的Pendrin蛋白。激光共聚焦顯微鏡拍照,觀察到COS-7細(xì)胞質(zhì)及細(xì)胞膜上均可觀察到綠色熒光蛋白的表達(dá),熒光表達(dá)較為清晰。在轉(zhuǎn)染S448X突變質(zhì)粒的COS-7細(xì)胞,在G418篩選10天左右細(xì)胞全部死亡未見能篩選到單克隆細(xì)胞。 4、表達(dá)SLC26A4野生型和S448X突變的細(xì)胞均能記錄到穩(wěn)定的氯離子電流,鉗制電壓變化時(shí),電流幅度隨之改變,具有電壓依賴性。統(tǒng)計(jì)學(xué)分析表明,在各個(gè)鉗制電壓下,兩者電流幅值有明顯差異(P0.05),而對(duì)照組未轉(zhuǎn)染的coS-7細(xì)胞記錄到與表達(dá)S448X突變的細(xì)胞相似大小的電流(p0.05)。給予氯離子阻斷劑NPPB作用20min后,表達(dá)SLC26A4野生型,S448X突變和未轉(zhuǎn)染細(xì)胞電流幅值均明顯減小(P0.05),說明三者電流均能被NBBP所抑制,這證實(shí)了記錄到的電流為氯離子電流。 5、表達(dá)SLC26A4野生型和S448X突變的細(xì)胞均能記錄到穩(wěn)定的鉀離子電流,統(tǒng)計(jì)學(xué)分析表明,在鉗制電壓30、50、70、90mV下,表達(dá)SLC26A4野生型COS-7細(xì)胞電流強(qiáng)度明顯強(qiáng)于表達(dá)S448X的cos-7細(xì)胞和未經(jīng)轉(zhuǎn)染的對(duì)照組COS-7細(xì)胞(P0.05)。給予的鉀離子通道離子阻斷劑TEACL作用20min后,表達(dá)SLC26A4野生型cos-7細(xì)胞,S448X突變coS-7細(xì)胞以及空白對(duì)照組細(xì)胞電流幅值均減低(P0.05)。通過分析Ⅰ-Ⅴ曲線圖,當(dāng)鉗制電壓在-90～10mV時(shí)各組鉀離子電流幅值均無明顯增加,各組間統(tǒng)計(jì)值無統(tǒng)計(jì)學(xué)差異(P0.05)；當(dāng)電壓在30-90mV時(shí),隨著膜電位去極化,各組鉀離子電流幅值明顯增加,Ⅰ-Ⅴ曲線向y軸靠近,表現(xiàn)出明顯的外向整流特性。 結(jié)論：免疫熒光化學(xué)發(fā)現(xiàn)野生型pendrin蛋白主要在細(xì)胞膜上表達(dá),突變體S448X主要表達(dá)在內(nèi)質(zhì)網(wǎng),膜片鉗實(shí)驗(yàn)顯示野生型離子轉(zhuǎn)運(yùn)能力強(qiáng)于突變體。本研究初步揭示了SLC26A4基因突變致聾的機(jī)理,即通過影響pendrin蛋白轉(zhuǎn)運(yùn),使其不能到達(dá)細(xì)胞膜形成陰離子通道,從而使陰離子轉(zhuǎn)運(yùn)受影響,同時(shí)SLC26A4基因突變可影響細(xì)胞外向整流鉀離子通道活性。這可能是SLC26A4基因突變導(dǎo)致耳聾和前庭導(dǎo)水管擴(kuò)大的重要原因。
[Abstract]:SLC26A4 mutation is an autosomal recessive inheritance. More than 200 different types of mutation have been found, which can lead to Pendred syndrome and DFNB4 (OMIM600791) non syndrome genetic deafness.Pendred syndrome. The clinical manifestations of the syndrome are goiter (normal thyroid function or slight decrease) and deafness caused by.SLC26A4 mutation in the deafness. The most common malformation is the enlargement of the vestibular aqueduct (enlarged vestibular aqueduct EVA) and the Mondini malformed.Pendrin in the inner ear, which mainly involved in the Cl-, the transport of HCO3-, which is related to the maintenance of the internal environment in the cochlea. In addition, the pendrin protein does not directly transfer the transmembrane transport of potassium ions, but the study found Pendrin to dimension. The internal potential of the cochlea (potassium ion balance potential) plays an important role in hearing loss. This study was based on the first SLC26A4 induced deafness mutation S448X, the constructed S448X mutants and the wild type SLC26A4 and GFP fusion gene expression plasmids, which were transferred to the dyed cells, and the Western blot was used to detect the protein expression, The green fluorescence of the fusion protein was observed directly and the immunofluorescence staining of the endoplasmic reticulum (ER), the Golgi body (Golgi), the microtubule tissue and the immunofluorescence method were used to observe the changes in the subcellular localization of the mutant pendrin protein. On the other hand, the whole cell current expressing the wild type and the mutant cells was recorded by the patch clamp technique. The effect of SLC26A4 gene mutation on chloride ion and potassium transport ability of cells was determined.
Objective: To observe the expression and functional changes of SLC26A4 gene and its deafness mutation S448X in vitro, and to explore the possible mechanism of its pathogenesis.
Methods: the mutant S448X and wild type SLC26A4 and EGFP fusion protein expression vector were constructed, COS-7 cells were transfected, the expression of protein was analyzed by Western blot, and the immunofluorescence staining of endoplasmic reticulum, Golgi body, microtubule tissue and the change of subcellular localization of mutated protein and wild type SLC26A4 were observed by laser confocal microscopy. Stable expression of mutant S448X and wild-type SLC26A4 cells were selected. The effects of mutant protein and wild type SLC26A4 on cell ion transport function were analyzed by whole cell patch clamp test.
Results: 1, Western blot was used to detect the expression of SLC26A4 wild type and S448X mutation and EGFP fusion protein in cOS-7 cells. Eukaryotic expression plasmid pEGFP N1SLC26A4S448X and pEGFP N1SLC26A4WT can be expressed in COS-7 cells. The size of the protein bands after expression is smaller than the wild type, indicating that the mutant protein is more wild than the wild type. The birth type is truncated.
2, pEGFP N1SLC26A4S448X in COS-7 cells, the green fluorescence is mainly distributed in the cytoplasm, and is Co located with the endoplasmic reticulum, and there is no co location with the Golgi bodies and microtubules, and the non mutant protein expression on the cell membrane is expressed in the cell membrane of the wild type protein of.PEGFP N1SLC26A4WT in the COS-7 cells, and a part of the green fluorescence is clustered in the cells. Quality.
3, the COS-7 cell line screened by G418 stably expressed the Pendrin protein encoded by wild type SLC26A4. The expression of green fluorescent protein could be observed on the cytoplasm of COS-7 cytoplasm and cell membrane, and the fluorescent expression was clear. The COS-7 cells transfected with S448X metamorphic granules were screened for 10 days in G418. All deaths were not found to be screened for monoclonal cells.
4, the cells expressing SLC26A4 wild type and S448X mutation can record stable chlorine ion current. When the voltage changes, the current amplitude changes and has voltage dependence. Statistical analysis shows that the current amplitude is significantly different under each clamp voltage (P0.05), while the non transfected coS-7 cells in the control group are recorded and expressed S4. 48X mutant cell similar size current (P0.05). After giving the chlorine ion blocker NPPB action 20min, the SLC26A4 wild type, the S448X mutation and the untransfected cell current amplitude decreased significantly (P0.05), indicating that the three current can be suppressed by NBBP, which confirms the recorded current as the chlorine ion current.
5, the cells expressing SLC26A4 wild type and S448X mutation can record stable potassium ion current. Statistical analysis shows that under the clamp voltage 30,50,70,90mV, the current intensity of SLC26A4 wild type COS-7 cells is stronger than that of the COS-7 cells expressing S448X and the untransfected control group COS-7 cells (P0.05). The potassium ion channel ions given by the cells are given. After the action of blocking agent TEACL for 20min, the expression of SLC26A4 wild type COS-7 cells, S448X mutation coS-7 cells and the blank control group were all decreased (P0.05). By analyzing the I-V curve, the amplitude of potassium current in each group was not significantly increased when the clamp voltage was -90 to 10mV, and there was no statistical difference between each group (P0.05). At 30-90mV, the amplitude of potassium current increased significantly with the depolarization of membrane potential, and the I-V curve approached to the Y axis, showing obvious outward rectifying characteristics.
Conclusion: the immunofluorescence chemistry showed that the wild type pendrin protein was mainly expressed on the cell membrane. The mutant S448X was mainly expressed in the endoplasmic reticulum. The patch clamp experiment showed that the wild type ion transport capacity was stronger than that of the mutant. This study preliminarily revealed the mechanism of the mutation induced deafness of the SLC26A4 gene, that is, it can not reach the pendrin protein transport by affecting the transport of the protein. The cell membrane forms anionic channel, which can affect the anion transport, and the mutation of SLC26A4 gene can affect the activity of the exportation potassium channel of the cell. This may be an important reason for the mutation of the SLC26A4 gene to cause deafness and the enlargement of the vestibular aqueduct.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R764.43

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3 編譯 張磊;MLC只是一個(gè)備選[N];中國(guó)計(jì)算機(jī)報(bào);2009年

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5 通訊員 唐德陽(yáng);中鐵四局集團(tuán)機(jī)電公司研制的砂漿車獲7項(xiàng)國(guó)家專利[N];人民鐵道;2010年

6 余海若;諾貝爾獎(jiǎng)為何６４次聚焦生命信使[N];大眾科技報(bào);2004年

7 古林;美國(guó)有哪些核彈頭？[N];中國(guó)國(guó)防報(bào);2002年

8 ;美白護(hù)膚又有新選擇[N];消費(fèi)日?qǐng)?bào);2001年

9 本報(bào)記者 林紫玉;網(wǎng)通成立全國(guó)性大客戶服務(wù)中心[N];通信產(chǎn)業(yè)報(bào);2002年

10 ;“大客戶”成為運(yùn)營(yíng)商業(yè)務(wù)重點(diǎn)[N];通信產(chǎn)業(yè)報(bào);2002年

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2 覃漢軍;前列腺癌趨化抗原基因疫苗研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);2005年

3 劉容枝;趨化抗原基因疫苗的研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);2006年

4 林倩;新生兒及嬰兒的聽覺特性與山東地區(qū)耳聾熱點(diǎn)基因突變及其聽力學(xué)表現(xiàn)的研究[D];山東大學(xué);2010年

5 賴若沙;極重度感音神經(jīng)性聾兒童致聾因素分析、常見基因檢測(cè)及其種族差異性研究[D];中南大學(xué);2011年

6 周逢海;縫隙連接介導(dǎo)的細(xì)胞間通訊及其與逼尿肌不穩(wěn)定關(guān)系的實(shí)驗(yàn)研究[D];第三軍醫(yī)大學(xué);2004年

7 韓冰;新生兒聽力及基因聯(lián)合篩查106,513例結(jié)果分析與技術(shù)研發(fā)及臨床意義研究[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2013年

8 司艷芳;RNA干擾cx43基因?qū)ε囵B(yǎng)的人RPE細(xì)胞的作用及其蛋白質(zhì)組學(xué)分析[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2008年

9 高蕾;人臍血源基質(zhì)細(xì)胞促進(jìn)巨核細(xì)胞增殖作用及機(jī)制探討[D];第三軍醫(yī)大學(xué);2008年

10 殷昊;植物嫁接體的發(fā)育：接口融合過程的時(shí)期劃分、表達(dá)譜分析及相關(guān)基因的鑒定[D];蘭州大學(xué);2012年

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1 張國(guó)正;中國(guó)人群特異的耳聾相關(guān)基因SLC26A4基因新變異致病性鑒定及其致聾機(jī)制研究[D];河北醫(yī)科大學(xué);2012年

2 王維;胃癌組織中SLC及其受體CCR7的表達(dá)與MMP-9、幽門螺桿菌L型感染的相關(guān)性研究[D];蚌埠醫(yī)學(xué)院;2011年

3 邊菁;人SLC真核表達(dá)載體的構(gòu)建，，表達(dá)及其效應(yīng)研究[D];華中科技大學(xué);2008年

4 楊小龍;中國(guó)西北地區(qū)藏族、土族、蒙古族非綜合征型耳聾常見聾病基因特征研究[D];蘭州大學(xué);2013年

5 魯峰;Fas介導(dǎo)的病理性瘢痕成纖維細(xì)胞死亡信號(hào)傳遞及基因調(diào)控的實(shí)驗(yàn)研究[D];第一軍醫(yī)大學(xué);2000年

6 董s

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