免疫脂质体介导的eNOS基因给药系统在高胆固醇血症内皮细胞中的表达

Expression of eNOS gene delivery system mediated by immunoliposome in hypercholesterolemic endothelial cells

目的 研究caveolin 1抗体介导的人内皮型一氧化氮合酶基因 -免疫脂质体给药系统在高胆固醇血症内皮细胞中的靶向转移与表达。方法 将体外培养的ECV30 4细胞分为正常组与高胆固醇血症组 ,每组分别用空载体、阳离子脂质体 eNOS (lip eNOS)、多聚赖氨酸修饰的caveolin 1抗体 lip eNOS复合物 (Ab lip eNOS)转染 ,同步设置不转染的阴性对照 ,实验设 3个复孔。转染后 4 8h ,用RT PCR检测细胞中eNOS基因mRNA水平 ,用黄递酶 NADPH活性染色法检测细胞中eNOS酶活性 ,用Griess法检测细胞培养上清液中NO含量。结果 Ab lip eNOS与lip eNOS能有效介导外源eNOS基因在正常与高胆固醇血症条件下内皮细胞中的表达 ,增加eNOSmRNA水平、eNOS活性与NO含量 ,与阴性对照和空载体组的内皮细胞中内源性eNOS基因的表达水平比较差异有显著性 (P <0 0 1) ,其中 ,Ab lip eNOS在高胆固醇血症条件下介导的eNOS基因表达效率最高 ,与Ab lip eNOS在正常条件下的表达效率、以及lip eNOS在正常与高胆固醇血症条件下的表达效率比较差异均有显著性 (P <0 0 1)。结论 Ab lip eNOS给药系统可促进外源eNOS基因在高胆固醇血症状态下内皮细胞中的表达 ,其机制可能与caveolin 1抗体介导的eNOS 免疫脂质体在细胞病变局部的靶向转移有关。

AIM To study the targeted transfer and expression of human endothelial nitric oxide synthase gene-immunoliposome delivery system mediated by caveolin-1 antibody in hypercholesterolemic endothelial cells. METHODS Cultured ECV304 cells were divided into normal and hypercholesterolemic groups and transfected with control vector pcDNA3.1, cationic liposome-eNOS complex (lip-eNOS) or polylysine modified caveolin-1 antibody-cationic liposome-eNOS complex (Ab-lip-eNOS) respectively. The negative control group was also used. The experiments were made in triplicates. 48 h after transfection, the cellular eNOS mRNA was detected by RT-PCR, the eNOS activity assayed by NADPH-diaphorase staining and the generation of NO in medium determined by Griess method. RESULTS Ab-lip-eNOS and lip-eNOS could efficiently induce the expression of exogenous eNOS gene in endothelial cells cultured in normal or hypercholesterolemic condition, increase eNOS mRNA level, eNOS activity and NO content, which were significantly different from the endogenous eNOS gene expression of negative control and control vector groups (P<0.01). And the expression efficiency of eNOS gene mediated by Ab-lip-eNOS was highest in hypercholesterolemia, which was significantly different from the gene expression by Ab-lip-eNOS in normal condition and the expression by lip-eNOS in both normal and hypercholesterolemia (P<0.01). CONCLUSION Ab-lip-eNOS delivery system could improve the expression of exogenous eNOS gene in hypercholesterolemic endothelial cells, and its mechanism might correlate with the targeted transfer of eNOS-immunoliposome mediated by caveolin-1 antibody in the cellular lesion.-