摘要
目的 建立大鼠肝窦内皮细胞 (SEC)的原代分离、培养方法 ,并研究其生物学特性。方法 胶原酶灌注结合Percoll密度梯度离心加选择性贴壁的方法分离SEC ;用光镜、扫描电镜观察培养SEC的形态学变化及超微结构 ;用Ⅷ因子和CD14免疫细胞化学染色及RECA 1单抗间接免疫荧光法观察SEC表面分子的表达。结果 分离培养的SEC得率为 (2 .0 6± 0 .35 )× 10 7/只大鼠 ,活力≥ 92 % ,纯度达 90 % ;光镜下细胞形态典型 ,SEM下可观察到特征性的窗孔结构 ;Ⅷ因子染色阴性而CD14染色阳性 ,RECA 1单抗间接免疫荧光染色阳性。结论 分离培养的SEC细胞得率、活力及纯度较高 ,形态典型且具有一般细胞所没有的窗孔结构及表面标志 ,为其功能和作用机制的深入研究奠定了基础。
Objective To set up a method for the primary isolation and cultivation of rat sinusoidal endothelial cells (SEC), and study their biological characteristics. Methods SEC were isolated by the combination of collagenase perfusion, percoll gradient isopycnic centrifugation and selective adherence; the changes on morphology and ultrastructure were observed under light microscope and SEM; the expression of surface antigens was measured by immunocytochemistry and immunofluorescence staining. Results The yield of the SEC after selective adherence was (2.06±0.35)×107/rat, the viability and purity was 92% and 90%. The isolated SEC have typical morphology and possess fenestrae. The SEC was positive for CD14 and negative for Ⅷ-factor related antigen. Immunofluorescence staining revealed that SEC were positive for RECA-1. Conclusions SEC isolated by this method provide a cell population not only with high purity and viability, but also with typical morphology, fenestrae and surface molecules.
出处
《消化外科》
CSCD
2004年第5期350-354,共5页
Journal of Digestive Surgery
基金
国家自然科学基金项目 (No .3 0 0 70 746)
关键词
肝窦内皮细胞
细胞培养
sinusoidal endothelial cell cell culture
