稳定表达人源GABAAR-CHO细胞株的建立

目的: 构建α₁亚基诱导表达、β₂和γ_2L亚基稳定表达的人源α₁β₂γ_2L-GABA_AR-CHO(Chinese hamster ovary)细胞株。方法: 从人cDNA文库中扩增α₁、β₂、γ_2L亚基编码基因,分别构建亚基表达载体;将三个亚基表达载体共转染CHO-K1细胞,通过抗性筛选、膜电位检测法进行稳定表达克隆筛选;通过qPCR、Western blot对亚基表达进行鉴定;以激动剂GABA、阳性变构调节剂地西泮(diazepam,Dia)、拮抗剂荷包牡丹碱(bicuculine)为工具药,采用全细胞膜片钳方法及膜电位检测法对稳定表达细胞的药理学功能进行鉴定。结果: 经克隆筛选获得表达量较高的α₁β₂γ_2L-GABA_AR-CHO并对其亚基表达鉴定,结果显示该细胞稳定表达α₁、β₂、γ_2L亚基,构建的α₁β₂γ_2L-GABA_AR-CHO细胞仅在加入四环素(tetracyclin)诱导的情况下表达α₁亚基并与β₂、γ_2L组装成具有功能活性的α₁β₂γ_2L-GABA_AR;对其进行全细胞膜片钳检测研究发现,GABA可对其产生激动效应,引起α₁β₂γ_2L-GABA_AR-CHO细胞产生氯离子通道特征性电流变化,Dia可剂量依赖性地增强GABA对α₁β₂γ_2L-GABA_AR的激动效应;在膜电位检测研究中,获得GABA激动效应EC₅₀为(177.72 ± 15.92)nmol/L,Dia变构效应EC₅₀为(3.63±0.52)μmol/L,拮抗剂Bicuculine拮抗效应IC₅₀为(538.83±29.55)nmol/L。结论: 通过采用诱导表达策略,成功构建了α₁β₂γ_2L-GABA_AR-CHO稳定表达细胞株,该细胞株具有对激动剂、阳性变构剂、拮抗剂特异性检测的药理学功能。

Establishment of Human GABAAR-CHO Cell Line Stable Expression

Objective: A functional α₁β₂γ_2L-GABA_AR-CHO human cell line is constructed in which α₁ subunit is inducd expression and β₂ and γ_2L subunits are stable expression. Methods: The coding genes of human subunit α₁, β₂ and γ_2L were amplified from human cDNA library, and the subunit vectors were respectively constructed. The three subunit vectors were cotransfected into CHO-K1 cells, and the stable expression clones were screened by resistance screening and membrane potential detection. The expression of subunits were identified by qPCR and western blot;the pharmacological function of α₁β₂γ_2L-GABA_AR-CHO line were identified by whole-cell patch clamp detection and membrane potential detection method. Results: The α₁β₂γ_2L-GABA_AR-CHO with high expression level was obtained by screening the clones. The cells stably expressed α₁, β₂ and γ_2L subunits. The constructed α₁β₂γ_2L-GABA_AR-CHO cells expressed α₁ subunit in the presence of tetracycline, and assembled with β₂ and γ_2L subunits to form α₁β₂γ_2L-GABA_AR with functional activity. Whole-cell patch clamp detection of α₁β₂γ_2L-GABA_AR-CHO showed that GABA could stimulate it and cause the characteristic current change of chloride channel in α₁β₂γ_2L-GABA_AR-CHO, and diazepam could enhance the activation effect of GABA on α₁β₂γ_2L-GABA_AR. Membrane potential detection showed that EC₅₀ of agonist GABA was (177.72±15.92) nmol/L, EC₅₀ of allosteric agent diazepam was (3.63±0.52) μmol/L, and IC₅₀ of antagonist bicuculine was (538.83±29.55) nmol/L, respectively. Conclusion: α₁β₂γ_2L-GABA_AR-CHO cell line is successfully constructed by the induced expression strategy, which has the pharmacological function of specific detection of agonists, positive allosteric agents and antagonists.