Prestin在Spag6基因敲除小鼠外毛细胞中的表达

Prestin expression in outer hair cells of Spag6 gene knockout mice

目的探究prestin在精子相关抗原6(Spag6)基因敲除小鼠中的表达及其与SPAG6之间的关系。方法获取小鼠耳蜗基底膜组织,进行免疫染色,采用共聚焦显微镜观察prestin与SPAG6在毛细胞中的共同分布。比较野生小鼠(Spag6+/+)、杂合型小鼠(Spag6+/-)和同窝基因敲除小鼠(Spag6-/-)外毛细胞prestin的荧光强度差异和毛细胞的形态改变,并以蛋白印记实验和实时定量聚合酶链反应检测3种基因型小鼠的prestin表达水平。结果SPAG6存在于野生型和杂合型成年小鼠的外毛细胞中,在表皮板处呈阳性表达,与prestin共同分布于外毛细胞的外侧壁处。基因敲除小鼠prestin的荧光强度较野生小鼠更弱,并且外毛细胞出现形态异常。蛋白印迹实验和实时定量聚合酶链反应均显示,基因敲除小鼠prestin表达量显著降低。结论SPAG6参与外毛细胞的构成,且影响外毛细胞prestin表达,提示两种蛋白之间存在相互作用。

Objective To detect the prestin expression in sperm associated antigen 6（ Spag6） gene knockout mice and its possible relationship with SPAG6. Methods Through a confocal microscope,the isolated basilar membrane was immunostained for observing the distribution of prestin and SPAG6 in hair cells. In particular,the fluorescence intensity of prestin and the morphological alterations of hair cells were compared among wild type mice（ Spag6 ＋ / ＋）,heterozygous mice（ Spag6 ＋ /-） and the corresponding Spag6 gene knockout littermates（ Spag6- /-）. The expressions of prestin protein and mRNA in mice of three genotypes were measured by Western blotting and real-time Q-PCR.Results SPAG6,which was positive at the cuticular plate and co-localized with prestin at the cell lateral wall,expressed in outer hair cells（ OHCs） of both wild type（ Spag6 ＋ / ＋） and heterozygous（ Spag6 ＋ /-） adult mice.Compared with wild type（ Spag6 ＋ / ＋） mice,gene knockout mice（ Spag6- /-） showed the lower fluorescence intensity of prestin and apparent morphological abnormity of OHCs. The expressions of prestin at protein and mRNA levels in gene knockout mice（ Spag6- /-） were significantly decreased as evidenced by Western blotting and real-timeQ-PCR. Conclusion SPAG6 participates in the formation of the cell membrane of OHCs and affects the expression of prestin in OHCs,which implies a possible interaction of these two proteins.