小鼠肝脏窦状内皮细胞分离和培养的一种新方法  

A new method for the isolation and culture of liver sinusoidal endothelial cells

作　　者：赵秀华[1] 罗彬[1] 罗盘[1] 程腊梅[1] 

机构地区：[1]中南大学生殖与干细胞工程研究所,长沙410078

出　　处：《国际病理科学与临床杂志》2010年第1期1-7,共7页Journal of International Pathology and Clinical Medicine

基　　金：国家重点基础研究发展计划项目(2007CB947900);湖南省自然科学基金重点项目(08JJ3075)~~

摘　　要：目的:建立小鼠肝窦状内皮细胞(liver sinusoidal endothelial cells,LSEC)的分离培养方法,研究其生物学特性。方法:中性蛋白酶消化小鼠肝脏组织,Percoll密度梯度离心分离消化后的细胞悬液,用内皮细胞筛选培养基及酶差异消化法纯化LSEC;免疫细胞化学染色检测LSEC的表面标志,分析LSEC的超微结构,观察DiI荧光标记的乙酰低密度脂蛋白(acetylated-low density lipoprotein,DiI-Ac-LDL)摄取能力,以体外成血管能力分析LSEC的功能。结果:分离纯化后的细胞呈典型鹅卵石样内皮细胞形态,这些细胞表达VIII因子,不表达CD31,CD90和CK19,具有窦状内皮细胞特征性的窗孔结构;能摄取DiI-Ac-LDL,并有一定的成血管能力。结论:建立了一种分离、培养LSEC的方法,为研究LSEC的功能奠定了基础。Objective To establish a method for the isolation and culture of mouse liver sinusoidal endothelial cells（LSEC）,and to study their biological characteristics.Methods Liver tissue from mouse was treated with a solution containing 2 U/mL dispase,and then the cell suspension was separated by Percoll density gradient centrifugation.LSEC was purified by culturing in an endothelial cell selective medium and passaged with enzyme discrepancy digestion.LSEC immunophenotypic characteristics were analyzed by using cyto-immunostaining.The ultrastructures of LSEC,the ability of uptaking the acetylated low-density lipoprotein（acetylated LDL）,and the vascular structures in vitro were detected.Results The isolated cells showed the typical cobblestone morphology characteristic of endothelial cells.These cells expressed factor Ⅷ but not CD31,CD90,and CK19.They were able to uptake the acetylated low-density lipoprotein and to form vascular structure.The results of electron microscope showed that these cells possessed typical transcellular fenestrations with a sinusoidal origin.Conclusion We develop a method for the isolation and culture of LSEC with typical morphologic and phenotypic characteristics.

关 键 词：肝窦状内皮细胞 条件培养基 窗孔 成血管结构 小鼠 

分 类 号：R575[医药卫生—消化系统]