人肝癌微血管内皮细胞黏附分子表达特点及其对外周血单个核细胞黏附和跨内皮迁移的影响

目的:研究人肝癌微血管内皮细胞(human liver cancer microvascular endothelial cell,HLCMVEC)上黏附分子表达的特点及其对免疫细胞黏附和迁移的影响.方法:分离培养HLCMVEC,以人肝窦内皮细胞系(liver sinusoid endothelial cell, LSEC)作为正常对照.通过细胞ELISA方法比较多种黏附分子在两种内皮细胞上的表达.外周血单个核细胞(peripheral blood mononuclear cell,PBMC)用荧光染料BCECF标记,将其与HLCMVEC或LSEC共培养,随后采用倒置荧光显微镜和连续光谱荧光仪检测PBMC在两种内皮细胞上的黏附和跨内皮迁移能力;此外,共培养前分别预加各种黏附分子的功能抗体,然后分析各种黏附分子在PBMC与肿瘤微血管内皮细胞黏附和迁移中的作用.结果:HLCMVEC表达CD31、CD34和细胞间黏附分子-3(intercellular adhesion molecule-3,ICAM-3)的水平低于LSEC(P＜0.05),表达ICAM-1和血管细胞黏附分子-1(vascular cell adhesion molecule-l,VCAM-1)的水平明显低于正常LSEC(P＜0.01),但表达整合素αvβ3和αvp5明显高于LSEC(P＜0.01).PBMC在HLCMVEC上的黏附和趋化剂诱导下的跨内皮迁移均显著低于LSEC[黏附:(205.5±46.0) vs (330.5±48.4)个,迁移:(49.0±10.6) vs(110.0±19.2)个,均P＜0.01];该黏附和迁移可被ICAM-1、ICAM-3、VCAM-1抗体明显阻断(P＜0.01),抗CD31抗体对黏附阻断不明显但能阻断跨内皮迁移(P＜0.05).结论:HLCMVEC特有的黏附分子表达特点抑制了PBMC的黏附和跨内皮迁移.

Expression characteristics of adhesion molecules on HLCMVEC and its influence on adhesion and trans-endothelial migration of PBMC

Objective:To investigate the expression characteristics of adhesion molecules on human liver cancer microvascular endothelial cells (HLCMVECs) and its influence on adhesion and trans-endothelial migration of immune cells.Methods:HLCMVECs were isolated and cultured,and human liver sinusoid endothelial cells (LSECs) were used as control.The expression of adhesion molecules was evaluated by cell-based ELISA.Peripheral blood mononuclear cells (PBMCs) were labeled with BCECF (a fluorescent dye) and then co-cultured with HLCMVEC or LSEC;the adhesion to endothelial cells and trans-endothelial migration of PBMCs were observed by inverted fluorescence microscope and continuous spectrum luminoscope.In addition,the functional antibody against each adhesion molecule was respectively added to endothelial cells before co-culture,which could determine the contribution of each adhesion molecule to the adhesion and trans-endothelial migration of PBMC.Results:Compared to LSECs,HLCMVECs expressed low level of CD31,CD34 and intercellular adhesion molecule-3 (ICAM-3) (P＜0.05) and an cven lower level of intercellular adhesion molecule-1 (ICAM-1) and Vascular cell adhesion molecule-1 (VCAM-1) (P＜0.01),but expressed significantly higher level of αv[β3 and αvβ5 (P＜0.01).Compared with LSECs,the adhesion of PBMCs to HLVMVECs were significantly decreased;and under the inducement of chemo-tactic agent,trans-endothelial migration of PBMCs through HLVMVECs were also significantly decreased (adhesion:[205.5 ± 46.0] vs [330.5 ± 48.4];migration:[49.0 ± 10.6] vs [110.0 ± 19.2];all P＜0.01).However,the adhesion and migration could be obviously blocked by antibodies against ICAM-3 (P＜0.05),ICAM-1 and VCAM-1(P＜0.01);Antibody against CD31 did not influence PBMC adhesion greatly but significantly reduced trans-endothelial migration (P＜0.05).Conclusion:The distinct expression of adhesion molecules on HLCMVECs reduced the adhesion and trans-endothelial migration of PBMCs.