局解手术学杂志2018，Vol.27Issue(1)：5-11,7.DOI:10.11659/jjssx.09E017036
大鼠原代肝星状细胞分离、鉴定及培养方法的研究
Isolation, identification and cultivation of primary rat hepatic stellate cells
摘要
Abstract
Objeetive To improve the method for the isolation and purification of rat hepatic stellate(HSC) cells and to provide a stable cell source for the research on liver-related diseases.Methods Rat liver was digested in situ by a two-step infusion assay under a strict control of the infusion temperature,flow rate and time with a combined utilization of Pronase E and Collagenase Ⅳ.And then,the HSC cells were separated by Percoll density gradient centrifugation.The cell growth curve and survival rate were measured by CCK-8 and trypan blue staining,respectively.The HSC cells were identified by flow cytometry and immunofluorescence cytochemistry.Results With the improved methods,there were (2.1 ± 0.2) × 107 HSC cells isolated from one rat and the survival rate was (96.2 ± 0.8) ％.The percentage of HSC cells with a spontaneous fluorescent characteristic from the isolated cells was 96.3％.The immunofluorescence cytochemistry was used to detect the expressions of the surface antigens α-SMA and Desmin in the isolated HSC cells.Conclusion By strict control of infusion temperature,flow rate and perfusion time as well as the combined application of Pronase E and Collagenase Ⅳ,there is an increased harvest of HSC cells with improved cell viability and purity,which is helpful for further research on HSC cells.关键词
肝星状细胞/细胞分离/原代培养/细胞鉴定Key words
hepatic stellate cell/cell isolation/primary culture/cell identification分类
医药卫生引用本文复制引用
张玉,周曦,易龙,马鑫,张乾勇,糜漫天..大鼠原代肝星状细胞分离、鉴定及培养方法的研究[J].局解手术学杂志,2018,27(1):5-11,7.基金项目
国家自然科学基金(81673155) （81673155）
重庆市科学技术委员会基金(cstc2017jcyjA0275) （cstc2017jcyjA0275）
重庆市研究生科研创新项目(CYS17295) （CYS17295）
重庆市自然科学基金(CSTC2017jcyjAX0275) （CSTC2017jcyjAX0275）
