低氧及一氧化氮对SW480细胞缺氧诱导因子-1α、VEGF及iNOS表达的初步研究

Effects of hypoxia and NO on the expression of HIF-1α, VEGF and iNOS in colon cancer cells SW480

目的:观察一氧化氮(NO)对低氧培养的人结肠腺癌细胞株SW4 80中缺氧诱导因子- 1α(HIF - 1α)、血管内皮生长因子(VEGF)、诱导型一氧化氮合酶(iNOS)表达的影响。方法:运用免疫细胞化学检测HIF - 1α、VEGF、iNOS蛋白表达,Westernblot检测HIF - 1α蛋白表达。原位杂交法检测HIF - 1αmRNA表达。结果:免疫细胞化学染色图像分析结果显示:低氧组细胞HIF - 1α、VEGF和iNOS蛋白表达水平显著高于常氧组(P <0 . 0 1,P <0 . 0 1,P <0 . 0 5 )和低氧+genistein(三羟基异黄酮)组(P <0 . 0 1,P <0 . 0 5 ,P <0 . 0 5 )。低氧条件下,SNP(硝普钠)显著抑制HIF -1α、VEGF蛋白的表达,但对iNOS表达无明显影响;NOC5 (NO发生剂)诱导HIF - 1α、VEGF、iNOS蛋白的表达;NO抑制剂L -NAME(N -硝基精氨酸甲酯)则抑制3者的表达。Westernblot结果显示:低氧培养条件下,HIF - 1α呈强表达,给予genistein能显著抑制其表达;而给予SNP和L -NAME后,蛋白表达量减少,给予NOC5 ,则蛋白表达增强。原位杂交结果显示:常氧组、低氧组及低氧+genistein组、低氧+SNP组、低氧+NOC5组、低氧+L -NAME组HIF - 1αmR NA表达A值组间均无显著差异。结论:低氧诱导SW4 80细胞HIF - 1α蛋白表达量增高,从而上调VEGF和iNOS表达。低氧条件下,NO供体SNP抑制SW4

AIM: To observe the expression of HIF-1α mRNA, HIF-1α, VEGF and iNOS proteins and to investigate their relationship in h ypoxia-treated SW480 cells. METHODS: HIF-1α, VEGF and iNOS proteins were measured by immuno cytochemistry. Western-blot was used to detect HIF-1α protein. HIF-1α mRNA wa s measured by in situ hybridization. RESULTS: Under hypoxic condition, SW480 cells expressed proteins of HIF-1α, VEGF and iNOS more strongly than that under normoxia condition. How e ver, under hypoxia condition, these three proteins expressed weakly or negativel y when the cells treated with genistein, the inhibitor of HIF-1α. Expressions o f HIF-1α and VEGF proteins in cultured SW480 cells under hypoxic condition were completely or partially inhibited by the addition of SNP but the expression of iNOS was unaffected. Another NO donor NOC5, however, induced the expression of t hese three proteins. L-NAME, a non-specific inhibitor of NOS, inhibited the expr ession of HIF-1α, VEGF and iNOS. The levels of HIF-1α mRNA changed slightly i n different oxygen condition or addition of genistein, NO donor or iNOS inhibitor . CONCLUSIONS: Hypoxia induces the expression of HIF-1α, therefor e upregulates the production of VEGF and iNOS. During hypoxia, SNP inhibits but N OC5 promotes HIF-1α expression, indicating that different NO donor acts on the cells through different mechanisms.