SLC26A4基因及其致聋突变在COS-7细胞中的表达与功能分析

发布时间：2018-07-29 19:41

【摘要】：SLC26A4突变所致疾病为常染色体隐性遗传,目前已发现200多种不同的突变类型,可导致Pendred综合征和DFNB4(OMIM600791)非综合征型遗传性耳聋。Pendred综合征临床表现为甲状腺肿大(甲状腺功能正常或轻微降低)和耳聋。SLC26A4突变导致的耳聋常常伴随有内耳畸形,其中最常见的畸形是前庭导水管扩大(enlarged vestibular aqueduct EVA)和Mondini畸形。Pendrin在内耳主要参与Cl-、HCO3-的转运,与维持耳蜗内环境稳定有关。另外,pendrin蛋白不直接调节钾离子的跨膜转运,但研究发现Pendrin对维持耳蜗内电位(钾离子平衡电位)产生听觉起到至关重要的作用。本研究以本实验室首先发现的SLC26A4致耳聋突变S448X,以及构建的S448X突变体和野生型SLC26A4与GFP融合基因表达质粒为基础,分别体外转染细胞,Western blot检测验证蛋白表达,然后直接观察融合蛋白绿色荧光,同时对内质网(ER),高尔基体(Golgi),微管组织免疫荧光染色,利用免疫荧光的方法观察突变体pendrin蛋白的亚细胞定位改变。另一方面,利用膜片钳技术记录分别表达野生型和突变体的细胞的全细胞电流的差异,明确SLC26A4基因突变对细胞氯离子和钾离子转运能力的影响。目的：观察SLC26A4基因及其一个致聋突变S448X在体外细胞中的表达与功能改变,以探讨其致病的可能机理。方法：将已构建完成的突变S448X和野生型SLC26A4与EGFP融合蛋白表达载体,转染cos-7细胞,Western印迹分析蛋白的表达,同时对内质网,高尔基体,微管组织免疫荧光染色,激光共聚焦显微镜观察突变蛋白和野生型SLC26A4的亚细胞定位改变。筛选稳定表达突变S448X和野生型SLC26A4细胞,全细胞膜片钳实验分析突变蛋白和野生型SLC26A4对细胞离子转运功能的影响。结果：1、Western blot检测SLC26A4野生型和S448X突变与EGFP融合蛋白在cOS-7细胞中的表达。真核表达质粒pEGFP N1SLC26A4S448X和pEGFP N1SLC26A4WT能在cos-7细胞中进行表达,表达后的蛋白条带大小与预期相符。S448X突变蛋白较野生型小,说明突变蛋白较野生型有截短。 2、pEGFP N1SLC26A4S448X在cos-7细胞中,绿色荧光主要分布在胞质内；并与内质网有共定位,与高尔基体、微管组织无共定位；细胞膜上无突变蛋白表达。pEGFP N1SLC26A4WT在cos-7细胞中野生型蛋白主要表达在细胞膜,可见部分绿色荧光聚集于细胞质。 3、G418筛选得到的COS-7细胞系稳定表达了野生型SLC26A4编码的Pendrin蛋白。激光共聚焦显微镜拍照,观察到COS-7细胞质及细胞膜上均可观察到绿色荧光蛋白的表达,荧光表达较为清晰。在转染S448X突变质粒的COS-7细胞,在G418筛选10天左右细胞全部死亡未见能筛选到单克隆细胞。 4、表达SLC26A4野生型和S448X突变的细胞均能记录到稳定的氯离子电流,钳制电压变化时,电流幅度随之改变,具有电压依赖性。统计学分析表明,在各个钳制电压下,两者电流幅值有明显差异(P0.05),而对照组未转染的coS-7细胞记录到与表达S448X突变的细胞相似大小的电流(p0.05)。给予氯离子阻断剂NPPB作用20min后,表达SLC26A4野生型,S448X突变和未转染细胞电流幅值均明显减小(P0.05),说明三者电流均能被NBBP所抑制,这证实了记录到的电流为氯离子电流。 5、表达SLC26A4野生型和S448X突变的细胞均能记录到稳定的钾离子电流,统计学分析表明,在钳制电压30、50、70、90mV下,表达SLC26A4野生型COS-7细胞电流强度明显强于表达S448X的cos-7细胞和未经转染的对照组COS-7细胞(P0.05)。给予的钾离子通道离子阻断剂TEACL作用20min后,表达SLC26A4野生型cos-7细胞,S448X突变coS-7细胞以及空白对照组细胞电流幅值均减低(P0.05)。通过分析Ⅰ-Ⅴ曲线图,当钳制电压在-90～10mV时各组钾离子电流幅值均无明显增加,各组间统计值无统计学差异(P0.05)；当电压在30-90mV时,随着膜电位去极化,各组钾离子电流幅值明显增加,Ⅰ-Ⅴ曲线向y轴靠近,表现出明显的外向整流特性。结论：免疫荧光化学发现野生型pendrin蛋白主要在细胞膜上表达,突变体S448X主要表达在内质网,膜片钳实验显示野生型离子转运能力强于突变体。本研究初步揭示了SLC26A4基因突变致聋的机理,即通过影响pendrin蛋白转运,使其不能到达细胞膜形成阴离子通道,从而使阴离子转运受影响,同时SLC26A4基因突变可影响细胞外向整流钾离子通道活性。这可能是SLC26A4基因突变导致耳聋和前庭导水管扩大的重要原因。
[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.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R764.43

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