EphA3基因稳定表达细胞模型的建立与分析

目的：建立稳定表达外源EphA3基因的小鼠成纤维细胞株模型,初步探讨EphA3基因表达对肿瘤发生、发展的影响。方法：通过脂质体介导的方法,将真核表达载体pcDNA3.1（-）/myc-his-EphA3转染NIH3T3细胞,用Western印迹确定外源EphA3基因表达;通过MTT实验、软琼脂集落形成实验,观察EphA3基因表达对NIH3T3细胞生物学特性的影响。结果：建立了稳定转染EphA3基因的NIH3T3细胞株;EphA3基因表达的小鼠成纤维NIH3T3细胞生长速度没有明显变化,但在软琼脂上锚着非依赖生长的能力加强。结论：建立了稳定表达外源EphA3基因的NIH3T3细胞株,EphA3基因稳定表达具有诱导正常NIH3T3细胞发生恶性转化的重要生物功能。

Construction and Analysis of EphA3 Gene Stable-Expressed NIH3T3 Cell Model

Objective： To establish an extraneous EphA3 gene stable-transfected mouse fibroblast cell line NIH3T3, and study the effect of EphA3 gene expression on tumor development and progression. Methods： The eukaryotic expression vector pcDNA3.1 （-）/myc-his-EphA3 was transfected into the mouse fibroblast cell line NIH3T3 by LipofeetAMINE. The integration and expression of extraneous EphA3 gene in NIH3T3 cells were analyzed by Western blot. The MTI＇, soft agar colony formation were used respectively to observed the effect of EphA3 gene expression on tumor development and progression in NIH3T3. Results： Extraneous EphA3 gene stably expressed mouse fibroblast cell line was successfully established. The integration and expression of extraneous EphA3 gene in NIH3T3 cells was confirmed by Western blot analysis. Comparing with the parental cells and the cells tranfected with empty vector, the EphA3 gene transfectants acquired the ability of growing on soft agar. Conclusion： Extraneous EphA3 gene stable expression in NIH3T3 cells promoted the proliferation of NIH3T3 cells in vitro and in vivo, causing the normal NIH3T3 cells malignancy transformation.