目的：观察SHIP1 对NSCLC细胞增殖的影响。方法：由NCBI Gene 数据库查询获得人SHIP1 基因CDS区，插至载体pTSB-CMV-MCS-SBP-3Flag-EGFP，构建SHIP1 真核过表达质粒；进一步利用其构建SHIP1 过表达慢病毒。用该慢病毒感染A549、SPCA-1 和PC-9 细胞株，获得SHIP1 稳定过表达NSCLC细胞系。Western blotting 和qRT-PCR分别从蛋白水平和mRNA水平检测SHIP1 的表达变化。采用MTT法和克隆形成实验检测过表达SHIP1 的PC-9 细胞增殖活力和克隆形成能力。Western blotting检测AP-1 蛋白复合体各组分的表达。结果：SHIP1 真核过表达质粒经测序证实构建成功。稳定过表达SHIP1 的A549、SPCA-1 和PC-9 细胞，在荧光显微镜下可见均一表达绿色荧光。与阴性对照组比较，细胞中SHIP1 在mRNA（P<0.01）及蛋白水平均明显升高。在PC-9 细胞中，过表达SHIP1 使细胞增殖、克隆形成能力降低（均P<0.01），p-c-Jun、FosB 等表达降低。结论：成功构建了稳定过表达SHIP1 的A549、SPCA-1和PC-9 细胞模型；过表达SHIP1可通过抑制AP-1 家族蛋白抑制NSCLC细胞增殖能力。

Objective: To observe the effect of SHIP1 on NSCLC cell proliferation. Methods: The CDS region of human SHIP1 gene was obtained by inquiring NCBI Gene database and was inserted into the vector pTSB-CMV-MCS-SBP-3Flag-EGFP to construct SHIP1 over-expression plasmid, which was further used to construct SHIP1 overexpression lentivirus. SHIP1 over-expressed lentiviruses were used to transfect A549, SPCA-1 and PC-9 cell lines to construct SHIP1 overexpressed NSCLC cell line. Western blotting and qRT-PCR were used to determine the protein and mRNA expression of SHIP1. The MTT assay and Clone formation assay were used to examine the cell proliferation ability and clone formation ability of PC-9 cells overexpressed SHIP1; Western blotting was performed to examine the level of AP-1 proteins. Results: The sequencing result suggested that the SHIP1 eukaryotic over-expression plasmid was successfully constructed. A519, SPCA-1 and PC-9 cells with SHIP1 over-expression were observed to display uniform green fluorescence under fluorescent microscopy. Compared with negative control group, the mRNA and protein levels of SHIP1 were significantly increased in SHIP1 overexpressed cells (all P<0.01). The over-expression of SHIP1 suppressed the abilities of proliferation and clone formation in PC-9 cells (all P<0.01), and down-regulated the expression of p-c-Jun and FosB etc. Conclusion: The SHIP1 overexpressed NSCLC cell lines were successfully established, and the over-expression of SHIP1 suppressed the cell proliferation ability by inhibiting AP-1 proteins in NSCLC cell lines.

国家自然科学基金资助项目（U1502222，No.81702295）；云南省卫生科技计划资助项目（No.2017NS177，No.2016NS113）；云南省科技计划资助项目（No.2014FB064）；中国博士后科学基金资助项目（No.2017M613008）