Tpr-Met转化的Ba/F3稳定株的构建

Construction of a recombinant stable Ba/F3 cell strain containing Tpr-Met

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。

AIM： To construct a stable cell strain encoding tumor-associated fused gene which expresses oncoprotein Tpr-Met. METHODS. We transfected Tpr-Met vector into Ba/F3 cells and screened the cell strain stably expressing Tpr-Met. The interleukin 3 （IL-3） independent proliferation of the cells was measured using the MTS assay. The expression of Tpr-Met, the activity of downstream signal transduction pathway and SU1 1274-induced inhibition of the signal pathway were investigated by Western blotting. RE- SULTS： We obtained a Ba/F3 cell strain stably expressing Tpr-Met. The cells presented IL-3 independent proliferation, suggesting a malignant transformation of the cell line. In Tpr-Met transformed Ba/F3 cells, the phosphorylation of Met and ERK were enhanced; however, specific c-Met inhibitor SU11274 suppressed the cell proliferation and c-Met phosphorylation. CONCLUSION： Tpr-Met transformed Ba/ F3 strain has been successfully constructed.