永生化人肝窦内皮细胞株的形态和功能特性研究

摘要

Objective To investigate the morphologic and functional characteristics of the immortalized human liver sinusoidal endothelial cell line(LSEC line).Methotis lmmunofluorescence staining and fluorescence microscopy were used to detect the classic endothelial cell markers in LSEC line,and flow cytometry was used to analyze the purity of the human LSEC line.The morphology(including W-P bodies and surface fenestrations)and phagocytotic capacity of the human LSEC line were observed by transmission and scanning electron microscope.The proliferation curve of the human LSEC line wag analyzed by MTT assay.The functional differences between the human LSEC line and human primary LSEC in expression of ELAM-1 and ICAM-1,activities of fibrinolysis(PAI-1,t-PA,u-PA),releasing of IL-6 and IL-8 were compared respectively bv enzyme linked immunosorbent assay.Comparison of the susceptibility to hypoxia-reoxygenation induced apoptosis between the human LSEC line and human primary LSEC were investigated by TUNEL Results The established human LSEC line maintained a high proliferative ability and has been passaged for more than 80 times in the absence of any growth factors.Immunofluorescence staning showed that the human LSEC line could express classic endothelial cell marks including von Willebrand Factor (vWF),and could take up acetylated low-density lipoproteins(Ac-LDL).The purity of the human LSEC line was confirmed over 95% by flow cytometric analysis.The W-P bodies and the phagocytosis of Dynabeads wag demonstrated by transmission electron microscope.And fenestrations could be found cellular surface with scanning electron microscopy.When compared with human primary LSEC,the human LSEC line hag an equivalent responsiveness to tumor necrosis factor in up-regulation of ELAM-1 and ICAM-1.The human LSEC line can also release PAI.t-PA.u-PA but can not release IL-6 and IL-8 to TNF-α.In contrast,human primary LSEC could release IL-6.The human LSEC line showed higher susceptibility to hypoxia-reoxygenation-induced apoptosis,and the percentage of apoptotic cells wag as high as(38.4±6.7)%.while(28.6±4.5)%and(7.8±1.2)%respectively in primary LSEC and in human umbilical vein endothelial cells.Conclusions The established human LSEC line maintains the special phenotypes and the major functional characteristics, and especially maintains the high susceptibility to hypoxia-reoxygenation-induced apeptosis.Therefore it is leasible to use this cell line for the study of liver ischemia-reperfusion injury.%目的 研究永生化人肝窦内皮细胞(LSEC)株的形态和功能特性,评价其作为体外研 究肝缺血再灌注损伤的细胞材料的可行性.方法 首先应用细胞免疫荧光染色、荧光显微镜以及流 式细胞术检测LSEC细胞株的纯度,透射和扫描电子显微镜技术观察细胞内的W-P小体和吞噬磁珠 的能力以及细胞表面窗孔结构.然后利用MTT法检测细胞的生长曲线,ELISA 法比较TNF-α刺激后人LSEC细胞株和原代LSEC黏附分子、纤溶活性分子和细胞因子的表达.最后采用TUNEL法比较两者对冷缺血再灌注损伤诱导凋亡的敏感性.结果 所建立的人LSEC 细胞株纯度达到95%以上.在形态上,人LSEC细胞株胞质内含有W-P小体,表面具有LSEC特异的窗孔结构.在功能上,人LSEC细胞株具有吞噬磁珠的能力和较强的增殖能力.在TNF-α刺激下,人LSEC细胞株和人原代 LSEC均剂量依赖性表达ELAM-1和ICAM-1,释放PAI-1、t-PA和u-PA;但人LSEC细胞株不产生IL-6 和IL-8.经冷缺血再灌注后,人LSEC细胞株的凋亡细胞百分率达到(38.4±6.7)%,明显高于人原代LBEC的(28.6±4.5)%和人脐静脉内皮细胞的(7.8±1.2)%.结论 已建立的人LSEC细胞株具有人原代LSEC的形态和主要功能特性,保留了对冷缺血再灌注损伤诱导凋亡的高度敏感性,因此可以作为体外研究肝脏缺血再灌注损伤机制的细胞材料.