STAT5b靶控报告基因检测胰岛素受体激酶活性细胞模型的建立

Establishment of cell model for determining activity of insulin receptor kinase by STAT5b responsive reporter gene

目的建立方便快捷的检测胰岛素受体激酶活性的方法,为筛选抗糖尿病的药物提供一种新的细胞模型。方法用克隆有胰岛素受体基因、STAT5b基因和STAT5响应元件靶控的荧光素酶报告基因的质粒共转染CHO细胞,RT-PCR检测外源基因的表达。转染的细胞经胰岛素处理,考察胰岛素对报告基因表达的剂量和时间效应,并采用AG1024处理和PTP1B基因共转染考察模型的特异性。结果经瞬时共转染的CHO细胞中检测到胰岛素受体和STAT5b基因表达,转染细胞中荧光素酶表达受胰岛素诱导且呈浓度依赖性和时间依赖性,最高诱导表达率为6.25倍。胰岛素受体酪氨酸激酶特异性抑制剂AG1024和酪氨酸磷酸化酶1B(PTP1B)可特异地阻断胰岛素的对荧光素酶表达的诱导效应。结论该细胞模型通过报告基因表达检测胰岛素受体激酶活性,灵敏性高、特异性强,在胰岛素受体激动剂和增敏剂的筛选方面具有应用价值。

Aim To develop a simple and rapid method to monitor insulin receptor kinase activity and provide a novel cell-based model for screening anti-diabetes drugs. Methods CHO cells were cotransfected by plasmids which respectively contained insulin receptor gene, STAT5b gene and lueiferase gene driven by STATS response elements. The expression of exogenous gene in transfected cells was examined bv RT-PCR. The transfected cellwere treated by insulin, and then the concentration and time-dependent response of luciferase expression to insulin induction was examined. Moreover, the specificity was identified by AG1024 treatment and PTP1B gene transfeetion. Results Expressions of insulin receptor and STATSb were detected in the transfected CHO cells. The expression of luciferase in transfected cells was induced by insulin in concentration and time-dependent way. The maximal induction fold was 6. 25. Moreover, the inducible expression of luciferase by insulin could be specifically blocked by tyrphostin AG1024, an inhibitor of insulin receptor kinase, or co-transfected PTP1B gene. Conclusions The insulin receptor kinase activity can be detected by expression of reporter gene with high sensitivity and specificity in this cell model, and with potential value in high throughput screening for insulin receptor activators and sensitizers.