CAJ | 학술논문

目的：建立大鼠肝窦内皮细胞（SEC）的分离培养的方法，观察SEC体外培养的生长特点。方法通过门静脉插管进行胶原酶原位灌注，结合离体消化、Percoll密度梯度离心法分离SEC；应用Ⅷ因子和CD14间接免疫荧光法观察SEC表面分子的表达进行细胞鉴定；应用光学显微镜、电子显微镜、并首次使用原子力显微镜（AFM）观察体外培养状态下SEC的超微结构及形态学变化。结果 SEC获得率为每只大鼠（2.95±0.31）×107，细胞活力好。免疫荧光法鉴定CD14阳性率约达99%，Ⅷ因子相关抗原抗体阳性率约为2%。光学显微镜下观察，细胞形态变化典型，并持续增殖。电子显微镜下观察到细胞典型的由成簇窗孔组成的筛板样结构；AFM下实现了对SEC的三维结构的观察，细胞骨架清晰。结论高纯度、活力好的SEC的成功培养及体外生长规律的观察，为研究SEC在肝脏生理病理过程中作用提供了前提条件。
목적：건립대서간두내피세포（SEC）적분리배양적방법，관찰SEC체외배양적생장특점。방법통과문정맥삽관진행효원매원위관주，결합리체소화、Percoll밀도제도리심법분리SEC；응용Ⅷ인자화CD14간접면역형광법관찰SEC표면분자적표체진행세포감정；응용광학현미경、전자현미경、병수차사용원자력현미경（AFM）관찰체외배양상태하SEC적초미결구급형태학변화。결과 SEC획득솔위매지대서（2.95±0.31）×107，세포활력호。면역형광법감정CD14양성솔약체99%，Ⅷ인자상관항원항체양성솔약위2%。광학현미경하관찰，세포형태변화전형，병지속증식。전자현미경하관찰도세포전형적유성족창공조성적사판양결구；AFM하실현료대SEC적삼유결구적관찰，세포골가청석。결론고순도、활력호적SEC적성공배양급체외생장규률적관찰，위연구SEC재간장생리병리과정중작용제공료전제조건。
Objective To establish a method for isolation and culture of hepatic sinusoidal endothelial cells (SECs) in rats , and to investigate the in?vitro growth characteristics of SECs. Methods SECs were isolated by in?situ collagenase perfusion through the portal vein,combined with in?vitro digestion and Percoll density gradient centrifugation. The expression of SEC surface molecules was subjected to cell identification by indirect immunofluorescence with factor Ⅷ and CD14. The optical microscopy,electron microscopy,and the first used atomic force microscopy (AFM) were employed to observe the ultrastructural and morphological changes of SECs under in?vitro culture. Results The SECs were obtained by(2.95 ± 0.31)× 107 cells from each rat,with good cell viability. The positive rate of CD14 reached approximately 99%as identified by immunofluorescence assay. The positive rate ofⅧfactor related antigen?antibody was about 2%. Under optical microscope,SECs showed typical morphological changes and persistent proliferation. The typical lamina cribrosa?like structure of the cells,composed of cluster fenestrae,was observed under the electron microscope. Visualization of three?dimensional structure of the SECs was achieved under AFM,with clear demonstration of cytoskeleton. Conclusion Successful culture of SECs with high purity and good vitality and demonstration of the in?vitro cell growth pattern can provide a basis for investigating the role of SECs in the pathophysiological process of the liver.