长春瑞滨联合西妥昔单抗抑制血管生成作用的实验研究

摘要

目的 探讨长春瑞滨联合西妥昔单抗对体内外血管生成的抑制作用.方法 以人肺腺癌细胞株A549为对照,采用四甲基偶氮唑蓝(MTT)法,观察长春瑞滨联合西妥昔单抗对人脐静脉内皮细胞(HUVEC)增殖能力的影响;应用Transwell小室模型、体外小管形成实验及流式细胞术,分别观察长春瑞滨联合西妥昔单抗对HUVEC迁移、小管形成及细胞凋亡的影响;应用鸡胚绒毛尿囊膜(CAM)模型,观察药物对体内CAM血管生成的抑制作用.结果 0.1、0.4和0.8 ng/ml长春瑞滨对HUVEC增殖的抑制率分别为25.8%、39.2%和54.0%,0.25 μg/ml西妥昔单抗对HUVEC增殖的抑制率为19.7%.0.1 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗和0.4 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗对HUVEC增殖的抑制率分别为29.5%和46.4%,联合用药有次加作用;0.8 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗对HUVEC增殖的抑制率为64.6%,联合用药有协同作用.0.1、0.4和0.8 ng/ml长春瑞滨分别联合0.25 μg/ml西妥昔单抗具有抑制HUVEC迁移的作用,抗HUVEC迁移率分别为51.9%、68.2%和95.0%,联合应用有协同作用.0.1 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗或0.4 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗对抑制HUVEC小管形成有次加或协同作用,抗HUVEC小管形成率分别为38.8%和57.7%;0.8 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗对抑制HUVEC小管形成有协同作用,抗HUVEC小管形成率为78.9%.0.8 ng/ml长春瑞滨+0.25 μg/ml西妥昔单抗诱导HUVEC的凋亡率为59.9%,有协同作用.长春瑞滨联合西妥昔单抗具有协同抑制CAM血管生成的作用.结论 小剂量长春瑞滨和西妥昔单抗在体内外具有抗血管生成作用,联合用药具有次加或协同作用.%Objective This experiment aims to study the anti-angiogenic ability of vinorelbine combined with cetuximab in vitro and in vivo.Methods Human lung adenocarcinoma A549 cells were used as control group.Proliferation of human umbilical vein endothelial cells(HUVEC)was assessed by MTT assay.Furthermore,we used Transwell chambers,capillary tube formation and flow cytometry to observe the effects of vinorelbine combined with cetuximab on HUVEC migration,tube formation and cell apoptosis,respectively.In addition,the anti-angiogenic ability of the drugs was checked using chicken chorioallantoic membrane(CAM)model.Results The inhibitory rate of HUVEC growth was 25.8%,39.2%,54.0% for vinorelbine at the concentration of 0.1 ng/ml,0.4 ng/ml,and 0.8 ng/ml,respectively;that of 0.25 μg/ml cetuximab was 19.7%,and that of 0.1 ng/ml vinorelbine + 0.25 μg/ml cetuximab,0.4 ng/ml vinorelbine +0.25 μg/ml cetuximab and 0.8 ng/ml vinorelbine + 0.25 μg/ml cetuximab was 29.5%,46.4%,64.6%,respectively.The inhibitory rates of the drugs at the above mentioned combinations of migration and tube formation of HUVEC were 51.9%,68.2%,95.0%,respectively.The inhibitory rate of 0.1 ng/ml +0.25 μg/ml cetuximab and 0.4 ng/ml vinorelbine +0.25 μg/ml cetuximab on tube formation of HUVEC was 38.8% and 57.7%,respectively,showing a sub-additive effect,and that of combination of 0.8 ng/ml vinorelbine +0.25 μg/ml cetuximab was 78.9%,showing a synergistic effect.In addition,the apoptotic rate of HUVEC induced by 0.8 ng/ml vinorelbine + 0.25 μg/ml cetuximab was 59.9%,showing a synergistic effect.The in vivo experiment also showed that the combination of the two drugs had a synergistic anti-angiogenic effect.Conclusion Both low dose vinorelbine and cetuximab have an anti-angiogenic effect in vitro and in vivo,and the combination of the two drugs has sub-additive or synergistic inhibitory effect on angiogenesis.