骨桥蛋白基因真核表达载体构建及对结肠癌细胞的作用

目的：构建骨桥蛋白（Osteopontin,OPN）基因真核表达载体,转染人结肠癌细胞SW480,分析其对SW480细胞株增殖及生存能力的作用。方法：构建重组表达载体pEGFP-N1/OPN,经测序鉴定无误后,转染人结肠癌SW480细胞。RT-PCR检测SW480细胞转染后OPN mRNA表达;Western blot检测SW480细胞转染后OPN蛋白表达量;Cell Counting Kit-8（CCK-8）测定细胞增殖;软琼脂克隆形成实验观察细胞的锚定非依赖性生长能力。结果：pEGFP-N1/OPN转染SW480后,OPN基因获得很好转录,OPN蛋白表达增高,转染OPN后SW480细胞增殖率（光吸收值）显著高于转染阴性组（P〈0.05）,软琼脂克隆形成速度增快,数量明显多于对照组和空载体组（P〈0.05）。结论：OPN基因真核表达载体pEGFP-N1/OPN构建成功,OPN具有促进SW480细胞增殖、存活的作用,为进一步研究OPN作用机制奠定了重要基础。

Construction of human osteopontin eukaryotic expression vector and its effects on proliferation and survival on colon cancer cells

Objective： To evaluate the effects of osteopontin （OPN） on proliferation and survival of SW480 colon cancer cells in vitro by constructing an osteopontin （OPN） eukaryotic expression vector. Methods： OPN gene was cloned into the eukaryotic expression vector pEGFP-N1. After sequencing, the vector was transfeeted into SW480 cells to analyze the OPN mRNA and protein expression by RT-PCR and Western blot, respectively. The impact on proliferation of cells were investigated by CCK-8 method, anchorage-independent growth was measured using soft agar assay. Results. The levels of OPN mRNA and protein of SW480 cells transfected with pEGFP-N1/OPN were distinctly increased. The OD450 value of transfection group was higher than negative control group （ P 〈0.05）. The anchorage-independent growth ability of cells with OPN transfection was also significantly increased in vitro （ P 〈0. 05）. Conclusion： In SW480 colon cancer cells, OPN was involved in the regulation of proliferation and survival.