Tpr-Met致NIH3T3细胞恶性转化分子机制浅谈.docVIP

Tpr-Met致NIH3T3细胞恶性转化分子机制浅谈 　 作者：郑红，汪思应，杨晓明，陈志武，李菲菲，倪芳 【摘要】 ［目的］ 探讨NIH3T3细胞恶性转化的分子机制。［方法］ 用表达Tpr-Met的重组质粒转染NIH3T3细胞致其恶性转化，用软琼脂集落实验和3H-TdR掺入DNA的方法检查细胞的生长状态及增殖性，用Western blot检测其c-Met及P-Erk、P-Akt表达水平，用EMSA实验检测NF-кB的结合活性。［结果］ 转染Tpr-Met的细胞形态有明显改变，能在软琼脂中形成集落，形成的集落大且明显增多，转染pcDNA3.1/c-Met/Tpr-Met的细胞集落数分别是0，71±10和160±12，说明Tpr-Met能诱导NIH3T3细胞恶性转化。用3H-TdR掺入DNA的方法来检测细胞的增殖性，发现转染Tpr-Met的 NIH3T3细胞和转染c-Met的NIH3T3细胞相比，转染Tpr-Met的细胞增殖能力明显增强，差异有极显著性（P＜0.01）。转化后的NIH3T3细胞DNA与NF-кB的结合能力明显增强，并且P-Erk和P-Akt的表达水平上调。［结论］ NF-кB参与了Tpr-Met致NIH3T3细胞恶性转化的信号调控，提示阻断该信号途径有可能抑制肿瘤的生长和转移。 【关键词】 恶性转化 Molecular Mechanism of Tpr-Met-Mediated Malignant Transformation in NIH3T3 Cells Abstract: ［Purpose］ To explore the molecular mechanism of NIH3T3 cells malignant transformation induced by Tpr-Met, the mutant of the c-Met receptor. ［Methods］ The human Tpr-Met gene was cloned into pcDNA3.1 eucaryotic expression vector. The recombinant plasmid and the control pcDNA3.1 vector were introduced into NIH3T3 cells by lipofectin-mediated gene transfection. Then positive colonies were selected by G418 and were tested for their proliferation by colony forming and incorporation of 3H-TdR. The DNA binding activity (nuclear transposal) of NF-кB was detected by electrophoretic mobility shift assay (EMSA), and c-Met, P-Erk, P-Akt protein expression were detected by Western blotting. ［Results］ Comparing with normal cells and c-Met-transfected NIH3T3 cells, the Tpr-Met-transfected NIH3T3 cells had changed evidently in cytomorphology, its ability of forming cell colonies in soft agar(0，71±10 and 160±12, respectively, Plt;0.01) increased, which revealed the malignant transformation of NIH3T3 cells induced by Tpr-Met. Taking incorporation of 3H-TdR as the index of proliferation, the ability of cell proliferation of the Tpr-Met-transfected NIH3T3 cells increased significantly. In the transformed cells, the expressions of P-Erk and P-Akt were upregulated at the protein level and the DNA binding activity (nuclear transposal) of NF-к