小剂量长春瑞滨抗血管生成化疗的实验研究

Experimental study of low doses of Vinorelbine in inhibiting angiogenesis

摘要:

目的：探讨长春瑞滨在体内外的抗血管生成活性及其可能的作用机制。方法：〖HTSS〗以人肺腺癌A549细胞为对照，采用MTT法、Transwell小室趋化实验、体外小管形成实验及流式细胞术（Flow cytometry,FCM），观察长春瑞滨对人脐静脉内皮细胞（Human umbilical vein endothelia cell,HUVEC）细胞增殖、迁移、形成毛细网管样结构的能力及细胞凋亡的影响；利用鸡胚绒毛尿囊膜（Chicken chorioallantoic membrane,CAM）模型，观察长春瑞滨对体内CAM新生血管的抑制作用。〖HTK〗结果：〖HTSS〗小剂量（0.1～1.0 ng/ml）长春瑞滨作用48 h，对HUVEC和A549细胞增殖抑制具有差异细胞毒作用（P=0.000）。0.1～0.8 ng/ml长春瑞滨对HUVEC作用12 h的迁移抑制率在20.56%～57.87%之间，与对照组相比，细胞迁移数明显减少（P<0.05,F=72.979,P=0.000）。0.1～0.8 ng/ml长春瑞滨对HUVEC作用24 h的小管形成抑制率在29.07%～56.83%之间，与对照组相比，小管形成数目明显减少且管腔结构趋于不完整，差异具有统计学意义（P<0.05,F=75.794,P=0.000）。0.1～0.8 ng/ml长春瑞滨作用48 h，诱导HUVEC细胞凋亡率在22.30%～37.05%之间，高于对照组细胞凋亡率15.60%（P<0.05；注：与对照组相比，在0.1、0.4、0.8 ng/ml长春瑞滨组的相应P=0.036、P=0.013、P=0.012）。体内实验显示，0.1～0.8 ng/ml长春瑞滨能够抑制CAM新生血管形成，抑制率在21.05%～42.11%之间，且抑制作用随药物浓度增加而增强。结论：①小剂量长春瑞滨在体外具有抑制血管内皮细胞增殖、迁移、小管形成及诱导细胞凋亡的作用。②小剂量长春瑞滨在体内具有抑制CAM血管形成的作用。

Abstract:

Objective:To investigate the antiangiogenic ability and mechanism of Vinorelbine in vitro and in vivo. Methods: In vitro, MTT colorimetric assay was used to observe the effect of Vinorelbine on the proliferation of human umbilical vein endothelial cell (HUVEC) and human pulmonary adenocarcinoma A549 cells. Transwell cabin test and out body canaliculization test were used to observe the impact of Vinorelbine on cell migration and capillary like tube formation ability, and the apoptosis rate of HUVEC was calculated by flow cytometry (FCM). In vivo, the chicken chorioallantoic membrane (CAM) model was used to check whether the neovascularization of CAM could be suppressed. Results: Low dose Vinorelbine (0.1～1.0 ng/ml) could inhibit the proliferation of A549 and HUVEC after 48 h treatment, and the inhibition had significant differences (P=0.000). The number of migrating HUVEC was decreased as compared with that in the control group (P<0.05,F=72.979,P=0.000), and the inhibition rate of migration was between 20/56%~40.40% when treated with 0.1~0.8 ng/ml Vinorelbine for 12 h. The number of tubules was reduced and the lumen lost its integrity as compared with that in the control group (P<0.05,F=75.794,P=0.000), and the inhibition rate of tube formation was between 29.07%~56.83% when treated with 0.1~0.8 ng/ml Vinorelbine for 24 h. When HUVEC was incubated with 0.1~0.8 ng/ml Vinorelbine for 48 h, the apoptosis rate was between 22.30%~37.05%. At the same time, the apoptosis rate was higher than that of 15.60% in the control group (P<0.05); Note: compared with that of the control, P value for 0.1,0.4 ng/ml, and 0.8 ng/ml Vinorelbine was P=0.036, P=0.013, and P=0.012 respectively). We also observed that Vinorelbine, at concentrations of 0.1, 0.4 ng/ml, and 0.8ng/ml, suppressed the neovascularization of CAM in vivo, and the inhibition rate was between 21.05%~42.11%. Moreover, the inhibition rate was enhanced as the drug concentration increased. Conclusion: ① In vitro, low dose Vinorelbine can inhibit the cell proliferation, migration, and tube formation of HUVEC, and has the ability of inducing apoptosis of HUVEC. ② In vivo, low dose Vinorelbine demonstrates antiangiogenic ability of CAM.