基于报告基因的胰岛素受体蛋白酪氨酸激酶激动剂细胞筛选模型的建立

目的建立用于大规模筛选胰岛素受体蛋白酪氨酸激酶(IRTK)激动剂的细胞模型。方法将含有编码胰岛素受体(IR)基因、转录激活因子5B(STAT5B)基因和STAT5应答元件调控的荧光素酶基因的质粒瞬时共转染仓鼠卵巢(CHO)细胞,人肝癌细胞HepG2和小鼠骨骼肌成肌细胞C2C12,将具有较高的胰岛素诱导表达荧光素酶活性的细胞经G418筛选,获得具有胰岛素诱导表达荧光素酶活性的细胞克隆。RT-PCR检测外源基因的表达的稳定性。荧光素酶活性分析和MTT法检测胰岛素处理后荧光素酶诱导表达的量效关系,以及AG1024和AG490处理后胰岛素诱导荧光素酶表达的特异性。并在96孔板上优化荧光素酶的检测条件。结果共转染的CHO细胞较HepG2和C2C12表现出更高胰岛素诱导的荧光素酶活性,经G418筛选得到一株具有较高诱导表达率的细胞克隆。RT-PCR表明,该细胞克隆表达外源胰岛素受体和STAT5B。该细胞中荧光素酶的诱导表达对于胰岛素具有剂量依赖性。IRTK抑制剂AG1024能阻断胰岛素诱导荧光素酶的表达,而Jak激酶抑制剂AG490无此作用。在96孔板上的荧光素酶的优化检测条件为:接种量每孔15×103个细胞,受试物诱导表达8h,Promega荧光素酶检测试剂按推荐量进行4倍稀释使用。结论在转基因CHO细胞中,IRTK的激活能灵敏、特异地诱导荧光素酶的表达。因此,该细胞模型可用于IRTK激动剂的高通量筛选。

Construction of cell model based on reporter gene for identifying agonist of insulin receptor protein-tyrosine kinase

AIM To develop a novel cell model for high throughout screening insulin receptor tyrosine kinase (IRTK) activators. METHODS Different cell lines including CHO, HepG2 and C2C12 were transiently transfected with plasmids which respectively contained insulin receptor gene, STAT5b gene and luciferase gene driven by STAT5 response elements. The transfected cell line which exhibited higher induced luciferase expression by insulin was placed under G418 pressure to produce and select clones of insulin responsive luciferase activity. Expression stability of exogenous gene was examined by RT-PCR. The concentration-dependent response of luciferase expression to insulin induction, and specificity of luciferase expression induced by insulin after pre-treated with AG1024 or AG490 were identified by lucferase expression assay and MTT assay. The assay condition for luciferase activity was also optimized on 96 well plate. RESULTS Co-transfected CHO cell exhibited higher insulin responsive luciferase activity than HepG2 and C2C12 cells. Under G418 pressure selection, a cell clone of high insulin responsive luciferase expression was isolated from the transfected CHO cell. In this clone, strong expression of exogenous insulin receptor and STAT5b was identified, and expression of luciferase was induced by insulin with concentration-dependent manner. Moreover, inducible expression of luciferase by insulin could be specifically blocked by tyrphostin AG1024, an inhibitor of insulin receptor kinase, but not by AG490, an inhibitor of Jak kinase. The optimal assay conditions for luciferase activity on 96 well plate were defined to seed cells 15 000 per well, induced for 8 h by test substances, and detected luciferase with quarter of reagent amount recommended by Promega kit. CONCLUTION Activation of IRTK could induce the luciferase expression with high sensitivity and specificity in transgenic CHO cell clone. Consequently, the cell model can be applied in high throughput screening for IRTK activators.