血管内皮生长因子过度表达促进恶性黑素瘤细胞增殖机制研究

Mechanism of malignant melanoma cells' proliferation enhanced by over-expression of vascular endothelial growth factor

目的:研究血管内皮生长因子(VEGF)过度表达促进恶性黑素瘤(MM)细胞增殖的机制。方法:通过电穿孔法将VEGF165cDNA转染至MM细胞系A375中,细胞计数法、MTT法和酶联免疫吸附实验(ELISA)法分别检测A375细胞体外增殖效应和其分泌的VEGF蛋白水平,化学比色法和蛋白印迹法(Western Blot)分别检测转染前后的A375细胞合成的诱导型、内皮型、神经型一氧化氮合酶(iNOS、eNOS、nNOS)的活性和蛋白表达。结果:转染VEGF165cDNA后,A375细胞明显增殖,其分泌的VEGF在转染后72 h、96 h明显增加(P<0.01),其合成的iNOS的活性和蛋白表达在转染后48 h、72 h、96 h明显升高(P≤0.05),但在转染前后未检测到eNOS和nNOS的活性及蛋白表达,一氧化氮合酶抑制剂L-NAME呈剂量依赖性地抑制转染72 h后的A375细胞增殖活性。结论:iNOS可能在VEGF过度表达促进MM细胞增殖中发挥重要作用。

Objective： To investigate the mechanism of malignant melanoma cells＇ proliferation enhanced by the over- expression of vascular endothelial growth factor （VEGF）. Methods： VEGF165 cDNA was transfected into malignant melanoma cell line A375 by electroporation. A375 cells＇ proliferation in vitro and VEGF protein level were detected by cells counting, MTT assay and ELISA respectively. The activity and protein expression of iNOS, eNOS and nNOS before and after transfection were measured by chemical chromatometry ,nethod and Western blot respectively. Results： The proliferation of A375 cells transfected with VEGF165 cDNA, was enhanced as compared with untransfected group. The expression of VEGF in the supematant of A375 cells transfected with VEGF165 was significantly increased at 72 h and 96 h（ P 〈 0.01 ）. The activity and protein expression of iNOS from A375 cells produced had the similar change with VEGF. However, the activity and protein expression of eNOS and nNOS were both under detectable level before and after transfeetion. Nitric oxide synthase inhibitor, N- nitro- L- arginine methyl estem （L - NAME） inhibited transfected A375 cells proliferation in a dose - dependent pattern after 72 h. Conclusion： iNOS might play roles in the proliferation of malignant melanoma cell line A375 which is enhanced by VEGF overe - xpression.