大鼠肝细胞与胰岛内皮细胞融合细胞系的建立及鉴定

Establishment and Identification of a Fusion Cell Line Produced by Electrofusion of Rat Hepatocytes and Islet Endothelial Cells

目的 通过电融合法构建由肝细胞系及原代内皮细胞融合而成的杂交细胞。方法 利用EMS诱变构建BRL-3A HGPRT缺陷型细胞;利用胆总管灌注法及胰酶消化法获得胰岛内皮细胞;利用CRY-3B细胞电融合仪及HAT筛选策略获得融合细胞;利用流式细胞仪对融合细胞的体积、细胞内颗粒密度及DNA含量进行检测;通过实时PCR及Western blotting检测融合细胞特异性基因的表达情况。结果 成功构建了一株由大鼠肝细胞系BRL-3A与原代大鼠胰岛内皮细胞融合而成的四倍体BRL-ies细胞。这株细胞既具有内皮细胞表型,表达CD31,又具有肝细胞特性,表达较高水平的谷氨酰胺合成酶。结论 肝细胞系BRL-3A可与原代内皮细胞融合形成较稳定的四倍体细胞,同时两者特征表型得以保留。

Objective To establish a new hybrid cell line from rat hepatocytes and primary endothelial cells by electrofusion.Methods BRL-3A hypoxanthine-guanine phosphoribosyltransferase-deficient cells were established by EMS.Islet endothelial cells were obtained by common bile duct perfusion and trypsin digestion.The hybrid cells were obtained by cell electrofusion and histone acetyltransferase screening.The volume,intracellular particle density,and DNA content of the hybrid cells were detected by flow cytometry.Specific gene expression in hybrid cells was detected by real-time PCR and western blotting.Results The BRL-ies tetraploid cell line was established by electrofusion of rat hepatocyte line,BRL-3A,and primary rat islet endothelial cells.This cell line possessed both endothelial cell phenotypes and hepatocyte characteristics,and expressed CD31 and a higher level of glutamine synthetase.Conclusion BRL-3A liver cells and primary endothelial cells are able to electrofuse to form a stable tetraploid cell line that retains some characteristic phenotypes of the parent cells.