功能性β1整合素-GFP融合蛋白稳定过表达肝癌细胞系的建立

Construction and characterization of stableβ1-integrin-GFP fusion gene overexpressing HCC cell lines

目的建立β1整合素-绿色荧光蛋白(GFP)稳定过表达人肝癌细胞系并检测其对各基质蛋白的黏附能力。方法构建β1整合素-GFP融合蛋白真核细胞重组表达质粒,并将其转染至人肝癌Hep3B细胞,建立稳定过表达β1整合素的肝癌细胞系。利用RT-PCR,Western印记,免疫沉降和荧光显微镜观察融合基因;检测转染细胞的增殖情况及对各基质蛋白的黏附能力。结果获得β1整合素-GFP融合蛋白稳定转染过表达Hep3B细胞系;转染的β1整合素能与内因性α1,α5,α6整合素亚型形成二聚体;转染β1整合素对细胞增殖无明显影响,但使细胞对各基质蛋白的黏附能力明显增高。结论建立了功能性β1整合素-GFP稳定过表达的人肝癌细胞系,为进一步研究β1整合素基因的功能提供了一个有效的细胞系。

Objective To establish a functional HCC cell line overexpressing β1-integrin-GFP fusion gene. Methods Eukaryotic expression vector encoding β1-integrin-GFP fusion gene DNA was constructed by PCR and transfected into the human HCC Hep3B cells, and the stable transfectants were isolated. The β1-integrin-GFP fusion gene was examined by RT-PCR, western blotting, immunoprecipitation and fluorescence microscopy. The proliferation and adhesion to extracellular matrix （ECM） proteins of these cells were determined. Results A HCC cell line stably overexpressing β1-in- tegrin-GFP fusion gene was established. The transfected β1-integrin could dimerize the endogenous α1, α5 and α6 integrin subunits. Meanwhile, β1-integrin transfection had no effect on hepatoma cell proliferation, but significantly promoted the adhesive capacity to ECM proteins such as laminin, fibronectin, collagen I and collagen IV etc. Conclusions The functional HCC cell fine stably over expressing β1-integrin-GFP fusion gene is successfully established and it can be used for the further biological study on the β1-integrin in hepatoma cells.