中国人常见GJB2基因突变蛋白亚细胞定位研究

目的研究中国人常见三种GJB2基因突变（c.235de1C,c.299-300delAT,and c.176-191de1（16）bp）的亚细胞定位和致聋机理。方法用MegaTran 1.0将GJB2野生型和三种突变的绿色荧光蛋白融合表达载体转染HEK293细胞,转染48h后用红色ER-TrackerTM复染细胞,激光共聚焦显微镜下观察结果。结果转染野生型质粒后可见细胞膜上出现点状或线状绿色荧光,三种突变融合蛋白质粒转染后均可见HEK293细胞质中有弥漫分布的绿色荧光,而胞膜上没有表达;转染突变质粒的细胞用ER-Tracker TM复染后,激光共聚焦显微镜下见红色和绿色几乎完全重叠,提示上述三种突变蛋白没有出现在细胞膜上,而是滞留在内质网中。结论中国人常见三种GJB2基因突变没有到达细胞膜上形成缝隙连接,而是滞留在细胞内质网中,提示过多的突变蛋白可能通过诱发内质网应激以及后续的内质网应激相关细胞凋亡导致耳聋。

Three Common GJB2 Mutations Causing Nonsyndromic Hearing Loss in Chinese Populations are Retained in Endoplasmic Reticulum

Objective To investigate the subcellular location of the protein products of threeGJB2 mutants（c.235de1C,c.299-300 delAT,and c.176-191de1（16） bp） and to explore the deafness mechanism caused by these GJB2 truncation mutations.Methods Mutant-eGFP fusion protein vectors were transfected into HEK293 cells by MegaTran 1.0.Transfected cells were double-stained by ER-Tracker and observed under a confocal microscope.Results Cells transfected withwild type gave characteristic punctuate patterns of GJs in the cell membrane.In contrast,c.235de1C,c.299-300 delAT,and c.176-191de1（16） bp mutant proteins were found to be trapped in the ER,and were therefore unable to form GJs in the membrane.Conclusion The three most common GJB2 mutations found in the Chinese populations,c.235delC,c.299-300delAT,and c.176-191de1（16） bp,cannot form gap junctons（GJs） in the plasma membrane.These mutant proteins were re-tained in the endoplasmic reticulum（ER）,suggesting that ER stress（ERS） and subsequent ERS-induced cell death may be responsible for hearing loss caused by these GJB2 truncation mutations.