恶性转化NIH3T3细胞中NFκB的表达

Expression analysis of NFкB in malignantly transformed NIH3T3 fibroblasts

目的探讨c-met突变体(Tpr-Met)诱导NIH3T3细胞恶性转化中的NFκB表达及意义。方法以空载体pcD-NA3·1做对照,用表达Tpr-Met的重组质粒转染NIH3T3细胞,筛选阳性克隆。做软琼脂集落实验,并提取细胞核蛋白做电泳迁移率改变分析(Electrophoretic mobility shift assay,EMSA),以检测NFκB与DNA结合活性。结果发现转染Tpr-Met后的NIH3T3细胞表型改变,并可在软琼脂中形成集落,EMSA结果发现DNA与NFκB结合能力增强。结论Tpr-Met可能激活了NFκB信号途径,提示此信号途径可作为抗肿瘤生长和转移的一个新的治疗靶点。

Objective To study the effects and role of NFκB expression in NIH3T3 fibroblasts transformed by onco- genic form of the receptor （Tpr-met）. Methods The plasmid, pcDNA3.1/Tpr-Met, containing Tpr-Met gene, was transfected into the NIH3T3 fibroblasts by lipofectamine, with the eukaryotic expression vector pcDNA3.1 as control. The soft-agar colony formation assay was performed to detect cell viability and proliferation, and nuclear protein was extracted from those cells after transfection for EMSA analysis of the DNA binding activity of NFκB. Results The pcDNA3.1/Tpr-Met-transfected NIH3T3 cells had a transformed phenotype, being able to form colonies in soft agar. Compared with cells transfected with control pcDNA3.1 vector, the DNA binding activity （nuclear transposal） of NFκB was increased by means of EMSA. Conclusion Tpr-Met may activate NFκB signaling pathway, suggesting that it can be a potential therapeutic target for inhibiting growth and metastasis of tumor.