| 黄楚琴 周问渠 蔡磊 洪玮 付欣 李冰.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[J].,2014,14(7):1225-1228 |
| 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 |
| Establishment of Rat Lung Alveolar Epithelial Cell Lines StablyExpressing of GCLC Promoter and Luciferase Reporter Gene |
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| DOI： |
| 中文关键词: 谷氨酰半胱氨酸合成酶催化亚单位(GCLC) 启动子 荧光素酶报告基因 稳定细胞株 |
| 英文关键词: GCLC (Glutamate-cysteine ligase catalytic subunit) Promoter Luciferase reporter gene Stable cell line |
| 基金项目:国家自然科学基金项目（30971166） |
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| 中文摘要: |
| 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-alpha刺激后，
能使荧光素酶活性上升，差异有统计学意义（P<0.05）；Rapamycin，GSH-EE 刺激后，荧光素酶活性显著下降（P<0.05）。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。 |
| 英文摘要: |
| Objective:To construct a rat typeⅡ alveolar epithelial cells L2 with stable expression of GCLC promoter and
luciferase reporter gene.Methods: The luciferase reporter vector PGL4.19 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
cells were stimulated by various factors for 6h, and then detected the luciferase activity.Results:The GCLC-LUC plasmid was successfully
constructed, and the L2 cell with stable expression of luciferase was successfully established. The luciferase activity increased
significantly after treating with TNF-alpha(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. |
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