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目的研究重组人肝再生增强因子(hALR)转染肝细胞系的生物学活性,为生物人工肝细胞反应器提供合适的细胞材料。方法本研究采用转染重组质粒pcDNA3.1(-)hALR的HepG2细胞,经G418筛选,在体外培养、传代后,进行Western blot免疫印迹实验及免疫荧光实验,检测细胞中hALR的表达,并与未转染重组质粒的HepG2细胞进行比较;采用ELISA方法,检测两组细胞培养液中hALR的分泌情况;使用流式细胞仪检测细胞中增殖细胞相关核抗原Ki-67,了解重组质粒转染前后HepG2细胞的增殖状况。结果转染重组质粒pcDNA3.1(-)hALR的HepG2细胞功能稳定,形态良好,细胞表达及分泌hALR较未转染重组质粒的HepG2细胞多,且随着培养时间的延长,细胞培养液中的hALR含量迅速升高,优于未转染重组质粒的HepG2细胞;转染重组质粒的HepG2细胞中Ki-67阳性细胞计数显著高于未转染重组质粒的HepG2细胞,说明转染重组质粒使肝细胞增殖活跃。结论本研究结果表明,前期研究构建的转染了hALR基因的肝细胞系能较高表达hALR,且细胞增殖活跃。
Objective To study the biological activity of recombinant human augmenter of liver regeneration (hALR) transfection in liver cell line and to provide a suitable cell material for biological artificial liver cell reactor. Methods In this study, HepG2 cells transfected with recombinant plasmid pcDNA3.1 (-) hALR were selected by G418 and cultured in vitro. After passage, Western blotting and immunofluorescence were used to detect the expression of hALR in cells. HepG2 cells without recombinant plasmids were compared. ELISA was used to detect the secretion of hALR in the two cell culture media. Flow cytometry was used to detect the Ki-67 of proliferating cells in the cells. Proliferation of HepG2 cells. Results HepG2 cells transfected with recombinant plasmid pcDNA3.1 (-) hALR had stable function, good morphology, more cells expressing and secreting hALR than HepG2 cells without recombinant plasmids, and with the extension of culture time, hALR increased rapidly, which was better than that of HepG2 cells without recombinant plasmids. The number of Ki-67 positive cells in HepG2 cells transfected with recombinant plasmids was significantly higher than that of HepG2 cells without recombinant plasmids. Cell proliferation is active. Conclusions The results of this study indicate that the hALR gene-transfected hepatocytes constructed in the previous study can express hALR with high cell proliferation.