重组人肝再生增强因子基因转染肝细胞系的增殖活性研究

摘要

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 investigate the effects of augmenter of liver regeneration on the proliferation of HepG2 cells, and its mechanism of action;to provide a suitable liver cell line for the bio-reactor of bio-artiifcial liver support system. Methods HepG2 cells transferred with expression vector pcDNA3.1(-)hALR were screened by G418. After in vitro culture and subculture, the expression of hALR were detected by Western blot and immunolfuorescence assay in two HepG2 cell lines transferred with or without expression vector. The secretion of hALR in the culture lfuid of the two HepG2 cell lines were detected by ELISA. Ki-67 in two HepG2 cell lines were detected by lfow cytometry to understand the proliferation of HepG2 cells transferred with or without expression vector. Results The function of HepG2 cell line transferred with expression vector pcDNA3.1(-)hALR was stable. HepG2 cells transferred with expression vector pcDNA3.1(-) hALR expressed and secreted more hALR than HepG2 cells, and with the extension of culture time, the content of hALR in the culture lfuid of HepG2 cells transferred with expression vector pcDNA3.1(-)hALR increased more rapidly than those of HepG2 cells. The Ki-67 positive cells in HepG2 cells transferred with expression vector pcDNA3.1(-)hALR were signiifcantly more than those of HepG2 cells, which indicated that the proliferation of HepG2 cells became more active by transfection of expression vector pcDNA3.1(-)hALR. Conclusion This study indicated that the HepG2 cells transferred with expression vector pcDNA3.1(-) hALR can express more hALR, and the proliferation of the cells was very active.