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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 study its biological characteristics. Methods: The liver tissues of mice were digested with neutral protease and the cell suspension was separated by Percoll density gradient centrifugation. The LSEC was purified by endothelial cell selection medium and enzyme digestion. The surface markers of LSEC were detected by immunocytochemical staining. The ultrastructure was observed and the DiI-Ac-LDL uptake ability of DiI was observed. The function of LSEC was analyzed by in vitro angiogenesis ability. RESULTS: The isolated and purified cells showed the typical pebble-like endothelial cell morphology. These cells expressed factor VIII, but did not express CD31, CD90 and CK19. They had the characteristic pore structure of sinusoidal endothelial cells. They could absorb DiI-Ac-LDL Have a certain ability to become blood vessels. Conclusion: A method of isolation and culture of LSEC was established, which lays the foundation for studying the function of LSEC.