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目的:建立一种可用于高通量筛选c-Met抑制剂的细胞模型。方法:采用电转染将Tpr-Met表达载体导入Ba/F3细胞中并筛选出能够稳定表达Tpr-Met的细胞株。而后对这种稳定株的白细胞介素3(IL-3)非依赖性增殖、Tpr-Met的表达及下游通路的活化、c-Met抑制剂SU11274对细胞增殖和信号通路的抑制作用进行全面评价。通过酶标仪检测细胞MTS吸光度值判断细胞的增殖水平,通过Western blot法检测Tpr-Met的表达及下游通路的活化、c-Met抑制剂SU11274对细胞信号通路的抑制作用。结果:获得稳定表达Tpr-Met的Ba/F3细胞株,该细胞株呈现IL-3非依赖性生长;能够表达持续活化的Tpr-Met;下游信号通路中关键分子Erk的磷酸化水平显著提升;c-Met特异性抑制剂SU11274通过抑制Tpr-Met磷酸化抑制下游信号通路的活化并抑制稳定株细胞增殖。结论:成功构建了Tpr-Met转化的Ba/F3细胞株。
Objective: To establish a cell model that can be used for high-throughput screening of c-Met inhibitors. Methods: Tpr-Met expression vector was transfected into Ba / F3 cells by electroporation and cell lines stably expressing Tpr-Met were selected. Subsequently, IL-3 independent proliferation, Tpr-Met expression and downstream pathway activation of this stable strain were evaluated, and the inhibitory effect of c-Met inhibitor SU11274 on cell proliferation and signaling pathway was evaluated comprehensively . The cell proliferation was determined by measuring the MTS absorbance by microplate reader. The expression of Tpr-Met and the activation of downstream pathway were detected by Western blot. The inhibitory effect of c-Met inhibitor SU11274 on cell signal pathway was detected. Results: The Ba / F3 cell line stably expressing Tpr-Met was obtained. The cell line showed IL-3 independent growth and expressed constitutively activated Tpr-Met. The phosphorylation level of key molecule Erk in the downstream signaling pathway was significantly increased. Inhibition of Tpr-Met phosphorylation by c-Met-specific inhibitor SU11274 inhibits the activation of downstream signaling pathways and inhibits stable cell proliferation. Conclusion: The Tpr-Met transformed Ba / F3 cell line was successfully constructed.