目的：研究桥接整合因子1（bridging intergrator 1，Bin1）基因过表达后对非小细胞肺癌细胞株H1975细胞周期的影响及其作用机制。方法：构建携带Bin1基因的CMV-MCS-GFP-SV40-Neomycin-Bin1质粒，并转染H1975细胞（Bin1+组），另设置空白质粒转染组（Bin1-组）及空白对照组（Ctrl组），利用RT-PCR和Western blotting分别检测3组细胞中Bin1在mRNA和蛋白质水平的表达情况。流式细胞术检测不同处理组H1975细胞周期的变化，Western boltting分别检测各组中AKT、mTOR磷酸化水平及细胞周期相关蛋白（周期蛋白D1、CDK4、Rb）的表达情况。结果：与Bin1-组、Ctrl组比较，Bin1+组H1975细胞中Bin1在mRNA、蛋白水平表达明显上调（均P<0.05）； H1975细胞阻滞在G1期\[（60.53±1.89）% vs（46.14±1.56）%、（47.33±2.07）%，均P<0.05\]； Bin1+组H1975细胞内p-AKT、p-mTOR表达下调（均P<0.05），AKT、mTOR表达变化无统计学差异（P>0.05）；周期蛋白D1、CDK4的表达量均明显下调(P<0.05)，Rb表达量明显增加（P<0.05）。结论：Bin1基因在H1975细胞株过表达后明显诱导细胞周期阻滞，其机制可能是通过抑制AKT-mTOR通路及其细胞周期相关蛋白实现的。

Objective:To study effect of bridging intergrator 1(Bin 1) gene over-expression on cell cycle of non-small cell lung cancer H1975 line cell, and its mechanism. Methods: CMV-MCS-GFP-SV40-Neomucin-Bin1 plasmid carrying Bin1 gene was constructed and was transfected into the H1975 line cell (Bin1+ group). The H1975 line cell (Bin1- group) transfected with empty plasmid and the H1975 line cell (Control group) were set up. Expressions of Bin1 mRNA and protein in the H1975 cell of the 3 groups were respectively detected by RT-PCR and Western blotting assays. Cell cycle changes of the H1975 cell in the various treatment groups were examined by flow cytometry assay. Phosphorylation level of AKT and mTOR as well as expression of cell cycle related proteins (cyclinD1, CDK4 and Rb) in the various treatment groups were detected by Western blotting. Results: Comparing with the Bin1- and control groups, expressions of Bin1 mRNA and protein in the Bin1+ group were obviously up-regulation (P<0.05) , cell cycle of the H1975 cell in the Bin1+ group was blocked in G1 phase (\[60.53±1.89\]% vs \[46.14±1.56\]% and \[47.33±2.07\]%, P<005), and in the H1975 cell of the Bin1+ group, expressions of p-AKT and p-mTOR were significantly down-regulation (P<0.05) , expressions of AKT and mTOR were not significantly difference (P>0.05) , expressions of cyclinD1 and CDK4 proteins were markedly down-regulation and expression of Rb protein was evidently increased (P<0.05). Conclusion: Over-expression of Bin1 gene in the H195 cell could obviously induce blocking of the cell cycle and its mechanism might be caused by inhibiting AKT-mTOR pathway and its cell cycle related proteins.

国家自然科学基金青年科学基金资助项目（No. 81201607）；河北省杰出青年基金资助项目（No. H2014206320）；河北省高层次人才培养项目资助（No. A201401040）