稳定表达大鼠GCLC启动子及荧光素酶报告基因细胞株的建立

目的:建立稳定表达大鼠谷氨酰半胱氨酸合成酶催化亚单位(GCLC)启动子及荧光素酶报告基因的大鼠肺泡上皮细胞株。方法:克隆大鼠GCLC上游5.9kb的启动子序列并构建重组报道载体PGL4.19-GCLC-LUC,转染到大鼠Ⅱ型肺泡上皮细胞L2,经G418筛选以获得单细胞抗性克隆并在传代过程中能稳定表达荧光素酶活性;检测细胞荧光素酶表达与细胞数量相关性;PCR检测稳定细胞株基因组已整合的插入片段;刺激因子刺激6h,检测稳定细胞株的反应性。结果:成功构建PGL4.19-GCLC-LUC;构建的稳定细胞株能稳定地表达荧光素酶,且与细胞数量正相关;PCR检测稳定细胞株目的片段稳定整合基因组;TNF-α刺激后,能使荧光素酶活性上升,差异有统计学意义(P0.05);Rapamycin,GSH-EE刺激后,荧光素酶活性显著下降(P0.05)。结论:成功构建稳定表达大鼠GCLC启动子及荧光素酶报告基因的细胞株,将为高通量药物筛选以及进一步研究GCLC基因的转录调控提供重要的细胞研究手段。

Establishment of Rat Lung Alveolar Epithelial Cell Lines Stably Expressing of GCLC Promoter and Luciferase Reporter Gene

Objective： To construct a rat type II alveolar epithelial cells L2 with stable expression of GCLC promoter and luciferase reporter gene. Methods： The luciferase reporter vector PGIA. 19 including rat GCLC 5.9 kb proximal regulatory sequence was constructed. Then the GCLC-LUC plasmid was transferred into L2 cells, and the GCLC-LUC cell clones were obtained by screening with G418. The positive clones stably expressing firefly luciferase were identified by detecting the luciferase activity. The stable GCLC-LUC ceils were stimulated by various factors for 6h, and then detected the luciferase activity. Results： The GCLC-LUC plasmid was successfu- lly constructed, and the L2 cell with stable expression of luciferase was successfully established. The luciferase activity increased significantly after treating with TNF-α （P〈0.05）, and decreased after treating with Rapamycin, GSH-EE （P〈0.05）. Conclusion： The L2 cells with stable expression of luciferase may be a good method for drug screening in further reaeach.