CAJ | 학술논문

目的肝脏非实质细胞主要包括肝星状细胞、肝窦内皮细胞和巨噬细胞,上述3种细胞在肝硬化、肝癌等疾病的发生和进展中起重要作用;本研究旨在建立上述3种肝非实质细胞的分离方法,培养并鉴定其纯度.方法采用体外酶灌注消化肝脏,将肝脏组织剪碎并进一步消化成单细胞悬液,密度梯度离心技术结合贴壁培养分离法分离肝脏非实质细胞,以锥虫蓝染色检测分离所得细胞的存活率,采用自发荧光、细胞形态观察、免疫荧光以鉴定所获得细胞.结果从大鼠肝脏分离出的3种非实质细胞纯度较高,大于90％;其中肝星状细胞、肝窦内皮细胞存活率在85％以上,可传代培养7 ～10代.结论本研究采用体外酶灌注消化法结合密度梯度离心技术,从大鼠肝脏成功分离肝星状细胞、肝窦内皮细胞和肝巨噬细胞,该方法相对简单、易操作.
목적 간장비실질세포주요포괄간성상세포、간두내피세포화거서세포,상술3충세포재간경화、간암등질병적발생화진전중기중요작용;본연구지재건립상술3충간비실질세포적분리방법,배양병감정기순도.방법 채용체외매관주소화간장,장간장조직전쇄병진일보소화성단세포현액,밀도제도리심기술결합첩벽배양분리법분리간장비실질세포,이추충람염색검측분리소득세포적존활솔,채용자발형광、세포형태관찰、면역형광이감정소획득세포.결과 종대서간장분리출적3충비실질세포순도교고,대우90％;기중간성상세포、간두내피세포존활솔재85％이상,가전대배양7 ～10대.결론 본연구채용체외매관주소화법결합밀도제도리심기술,종대서간장성공분리간성상세포、간두내피세포화간거서세포,해방법상대간단、역조작.
Objective To establish an effective method to isolate hepatic non-parenchymal cells including hepatic stellate cells (HSCs),sinusoidal endothelial cells (SECs) and macrphages respectivly.Methods The whole livers were removed from rats under sterile condition,then the livers were perfused with digestive enzyme in vitro and cut into species.The species were digested to generate single cells suspension at 37 ℃.By using the methords of Percoll gradient centrifugation and adherent culture,the single cells were isolated into HSCs,SECs,and macrophages.The cell activity was observed by the trypan blue excluding test,and the cell purity was identified using spontaneous fluorescence,immunofluorescence and cellular morphology.Results The purity of hepatic non-parenchymal cells was more than 90％,and the survival rate of HSCs and SECs was more than 85％.The isolated HSCs and SECs could be cultured with 7-10 passages.Conclusion This isolation method is easy and reliable to separate HSCs,SECs and macrophages from the liver of rats with a high purity.