稳定表达中国人常见GJB2基因突变细胞系的建立  

The Establishment of HEK293 Cell Line Stably Expressing GJB2 Mutation Common in Chinese

作　　者：张延平[1] 张晓强[1] 刘雅莉[2] 黄玮 孙玉蕊[1] 

机构地区：[1]中国人民解放军第309医院耳鼻喉科,北京100091 [2]河南省医学情报研究所,郑州450052 [3]河南省卫生厅,郑州450052

出　　处：《中国听力语言康复科学杂志》2011年第4期11-14,共4页Chinese Scientific Journal of Hearing and Speech Rehabilitation

基　　金：基金项目：全军”十一五”项目归国人员课题（06H048）国家自然科学基金面上项目青年基金（30700936）：中国人民解放军第309医院院内基金（LCB08MSl2、309-09-03）

摘　　要：目的将中国人常见GJB2基因突变c．235delC真核表达载体稳定转染入HEK293细胞，建立稳定表达人c．235delC的HEK293细胞系。方法将pcx26-EGFP和pcx26—235delc—EGFP转染HEK293细胞。24h后加G418筛选两周，使用FCM分选GFP阳性细胞，继续培养。蛋白免疫印迹检测GFP表达，激光共聚焦显微镜检测蛋白表达。结果蛋白免疫印迹显示稳定转染人pcx26－EGFP和pcx26—235delC--EGFP的HEK293细胞都能够检测到GFP的蛋白条带，显微镜下可见稳定表达基因的HEK293细胞普遍表达GFP。结论本研究成功构建了稳定表达中国人常见GJB2基因突变的HEK293细胞系，为今后的研究工作打下基础。Objectives To construct the HEK293 cell line stably expressing GJB2 mutation common in Chinese. Methods The pCx26-EGFP and pCx26-235delC-EGFP were transfected into HEK293 cells. Twenty-four hours later, the cells were diluted and G418 was added to them. FCM cell sorter was used to select positive GFP cells 2 weeks after transfection. Then, western blot and laser confocal microscope were applied to identify the expression of c.235delC. ResuZts The western blot results showed that GFP was expressed in the HEK293 cells transfected with pCx26-EGFP and pCx26-235delC-EGFP.The characteristic expression patterns of wild- type GJB2 in the cell membrane and c.235deIC mutation in the cytoplasm can be seen under the confocal microscope. Conclusion The HEK293 cell line stably expressing GJB2 mutation common in Chinese has been successfully established, thus being able to provide a new tool to further explore the mechanism of non-syndromic hearing impairment.

关 键 词：感音神经性耳聋 缝隙连接 突变 

分 类 号：R734.2[医药卫生—肿瘤]