目的: 研究外源性分化抑制因子3（Inhibitor of differentiation 3,Id3）基因表达对人肺腺癌细胞系A549细胞生长的抑制作用。方法：构建Id3和EGFP的融合基因表达载体pEGFP/Id3,采用脂质体转染技术将pEGFP/ Id3导入A549细胞。FCM分析转染细胞EGFP表达效率，荧光显微镜观察EGFP表达情况；半定量RTPCR、免疫细胞化学分析转染后A549细胞 Id3 mRNA和蛋白的表达。MTT法检测转染后A549细胞生长抑制情况，FCM分析A549细胞周期进程，Annexin V/7AAD和Hoechst33258染色检测转染后细胞的凋亡情况。结果:成功构建人Id3与EGFP融合基因表达载体pEGFP/Id3；荧光显微镜和FCM观察到EGFP的有效表达，转染48~72 h时EGFP表达率最高；Id3 mRNA及蛋白在转染后的A549细胞中能有效表达。转染后48 h和72 h，pEGFP/Id3转染组A549细胞生长受到明显抑制（P<0.01）；细胞周期分析表明，pEGFP/Id3转染组G0/G1期细胞显著高于对照组（P<0.05）; Annexin V/7AAD双染结果显示，pEGFP/Id3转染组细胞的早期凋亡率\[（10.67±2.60）%\]显著高于pEGFP组\[（3.39± 2.21）%\]和未转染组\[（2.35 ± 0.95）%\]（P<0.05）；Hoechst33258染色结果也证实pEGFP/Id3组A549细胞出现明显凋亡形态特征。结论:外源性Id3基因在肺腺癌A549细胞中的表达能抑制细胞增殖,并诱导细胞凋亡。

Objective:To investigate the inhibitory effect of inhibitor of differentiation 3 (Id3) on growth of human lung adenocarcinoma cell line A549. Methods: Recombinant eukaryotic expression vector pEGFP/Id3 was constructed and transfected into A549 cells by liposomemediated method. Expression of pEGFP/Id3 in A549 cells was analyzed by flow cytometry (FCM), fluorescence microscopy, semiquantitative RTPCR and immunocytochemistry. The growth inhibitory rate of A549 cells was examined by MTT assay; cell cycle change was evaluated by PI (propidium iodide) staining method. Cell apoptotic rate and nuclear morphology were detected by Annexin V/7AAD and Hoechst33258 staining. Results: The recombinant eukaryotic expression vector pEGFP/Id3 was successfully constructed. The expression of EGFP reached the peak 4872 h after transfection; the expresion of pEGFPtransfected group was higher than that of the pEGFP/Id3 group. RTPCR and immunocytochemistry staining showed that Id3 mRNA and protein were effectively expressed in pEGFP/Id3transfected A549 cells. The growth of cells in pEGFP/Id3 transfected cells was significantly inhibited 4872 h after transfection（P<0.01）. More cells were blocked in G0/G1 phase in pEGFP/Id3transfected group compared with pEGFP group（P<0.05）. Annexin V/7AAD showed that the apoptotic rate of pEGFP/Id3 group （\[10.67±2.60\]%）were significantly higher than those of the control group（\[2.35 ± 0.95\]%）and pEGFP group（\[3.39±2.21\] %）（P<0.05）. Hoechst33258 staining also showed that the cells in pEGFP/Id3 group had typical apoptotic morphology.Conclusion: Exogenous Id3 gene expression in A549 cells can induce cell growth inhibition and apoptosis of A549 cells.

江苏省六大人才高峰基金资助项目（No.050203D）。