稳定表达人水通道蛋白4的胚肾细胞系HEK293/AQP4的建立及应用  

Establishment and Evaluation of a Human Embryonic Kidney Epithelial Cell Line HEK293 / AQP4 Stably Express Human AQP4

作　　者：刘俊秀[1] 孙巧松[1] 丰岩清[1] 曾缨[1] 赖蓉[1] 陈曦[1] 黄帆[1] 

机构地区：[1]中山大学附属第一医院神经科,广东广州510080

出　　处：《中山大学学报（医学科学版）》2010年第6期786-791,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：教育部高等学校博士学科点专项科研基金新教师课题(200805581139);中山大学医科青年教师科研启动基金(2008011);广东省医学科研基金(B2009056)

摘　　要：【目的】建立一种可临床应用的并能稳定表达水通道蛋白4的胚肾细胞系HEK293/AQP4,用于检测视神经脊髓炎患者血清中的AQP4抗体。【方法】构建有AQP4基因的表达载体,利用脂质体将载体转入HEK293细胞,G418筛选,RT-PCR、间接免疫荧光检测稳定细胞系中水通道蛋白4的表达及分布。细胞间接免疫荧光检测20例NMO患者、20例MS患者、30例健康对照血清中的AQP4抗体,并同鼠脑间接免疫荧光检测法比较。【结果】限制性酶切消化、PCR、测序结果证明载体构建成功。间接免疫荧光证实稳定细胞系中有AQP4表达,且主要分布在细胞膜上。20例NMO患者血清中11例NMO-IgG阳性,18例AQP4抗体阳性,滴度在1∶60-1∶15360之间,而MS患者仅检测到1例阳性,健康对照未检测到阳性。【结论】成功构建稳定表达水通道蛋白4的胚肾细胞系HEK293/AQP4,并检测出国人NMO患者血清中存在AQP4抗体,且敏感性高于鼠脑法,可以有效的鉴别视神经脊髓炎和多发性硬化。【Objective】 To establish a clinically applicable assay with human embryonic kidney epithelial cell line HEK293 that can stably express human AQP4,and to investigate the performance of this assay to detect anti-AQP4 antibodies in neuromyelitis optica （NMO） patient sera. 【Methods】 The expression vector containing aquaporin-4 （AQP4） gene was constructed and transfected into HEK293 cells mediated by liposomes. After G418 filtration,the expression of AQP4 was detected by RT-PCR and indirect immunofluorescence staining. We tested masked serum samples from 20 NMO patients,20 patients with multiple sclerosis （MS）,and 30 healthy controls. And the seropositivities and titre were compared with the indirect immunofluorescence with a composite substrate of mouse tissues. 【Results】 Recombinant eukaryotic expression vector contained human AQP4 cDNA was constructed successfully,approved by restriction enzyme digestion and DNA sequencing. Indirect immunofluorescence staining showed that the HEK293 cells transfected with pCDNA3.1 （＋）-AQP4 could express human AQP4,mainly in the cell membrane. We found antibodies to AQP4 in 18 of 20 patients with neuromyelitis optica using this method,while 11 of 20 patients with NMO using the rat brain assay. The antibody titre ranged from 1 ∶ 60 to 1 ∶ 15 360. In contrast,the patient with MS only have one case positive for AQP4 antibody and healthy control showed negative for AQP4 antibody with both assays. 【Conclusion】 We successfully established HEK293 / AQP4 cell line which can expressed AQP4,and can be used to detect anti-AQP4 antibody present in the sera of patient with NMO. The newly developed anti-AQP4 antibody assay appears to have a higher sensitivity for NMO than theindirect immunofluorescent method using mouse brain tissues. It is expected to be a useful tool for the early diagnosis of NMO and distinguish NMO from MS.

关 键 词：视神经脊髓炎 NMO-IGG 水通道蛋白4( AQP4) HEK293 

分 类 号：R54[医药卫生—心血管疾病]