Tpr-Met致NIHT细胞恶性转化的分子机制初探.docVIP

　　Tpr-Met致NIH3T3细胞恶性转化的分子机制初探 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et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 binant plasmid and the control pcDNA3.1 vector ediated gene transfection. Then positive colonies ing and incorporation of 3H-TdR. The DNA binding activity (nuclear transposal) of NF-кB obility shift assay (EMSA), and c-Met, P-Erk, P-Akt protein expression paring al 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, alignant 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 portant modulating role in the NIH3T3 cell malignant transformation, or groetastasis. Key alignant transformation; NF-кB; molecular mechanism c-Met 受体是由c-Met原癌基因编码的一个190kD的异二聚体蛋白质，由胞外50kD的α亚单位和跨膜的145kD的β亚单位通过二硫键组成，结构上属Ⅱ型酪氨酸蛋白激酶受体，是一类有自主磷酸化活性的跨膜受体，具有胞外、胞内和跨膜结构域。c-Met能调节肝细胞生长因子(HGF)的生物学活性。HGF与c-Met结合后，激活受体发生自身磷酸化，启动多条信号通路，发挥多种生物学效应，调节细胞的增殖、分化、形态发生和侵袭运动等[1，2]。 HGF-c-Met信号转导通路广泛存在于各种细胞中，对多种组织器官的生长发育具有重要的生理调节功