3种抗氧化剂对亚砷酸钠染毒人膀胱上皮细胞血管内皮生长因子表达的拮抗作用研究

目的探讨抗氧化剂褪黑素(melatonin,MEL)、N-乙酰半胱氨酸(N-acetyl cysteine,NAC)、维生素C(Vitamin,VC)对砷诱导人正常膀胱上皮(SV-HUC-1)细胞血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的拮抗作用。方法将处于对数生长期的SV-HUC-1细胞暴露于含终浓度分别为0(对照)、0.5、1、2、4、8、10μmol/L亚砷酸钠的F12K完全培养基染毒24 h,或者含终浓度分别为4、10μmol/L亚砷酸钠的F12K完全培养基染毒0(对照)、4、12、24、48、72 h;联合暴露组在加入含终浓度分为4μmol/L亚砷酸钠的F12K完全培养基前30 min分别加入1%二甲基亚砜(DMSO)和抗氧化剂MEL、VC、NAC(终浓度分别为0.5、1、1 mmol/L),染毒24 h。分别采用Western blot法和RT-PCR法检测SV-HUC-1细胞VEGF蛋白和mRNA的表达水平。结果 10μmol/L亚砷酸钠染毒组SV-HUC-1细胞VEGF mRNA的表达水平高于对照组,差异有统计学意义(P0.05);且随着亚砷酸钠染毒剂量的升高,SV-HUC-1细胞VEGF mRNA的表达水平呈上升趋势。与对照组比较,4μmol/L亚砷酸钠染毒12、24、48 h及10μmol/L亚砷酸钠染毒24和48 h后SV-HUC-1细胞VEGF mRNA的表达水平均升高,差异有统计学意义(P0.05);且随着亚砷酸钠染毒时间的延长,各剂量组SV-HUC-1细胞VEGF mRNA的表达水平均呈先上升后下降的趋势。与对照组比较,4μmol/L亚砷酸钠+DMSO染毒组SV-HUC-1细胞中VEGF蛋白的表达水平均增加,差异有统计学意义(P0.05)。与4μmol/L亚砷酸钠+DMSO染毒组比较,亚砷酸钠+NAC染毒组SV-HUC-1细胞中VEGF蛋白的表达水平较高,差异有统计学意义(P0.05);而亚砷酸钠与MEL和VC联合染毒组SV-HUC-1细胞中VEGF蛋白的表达水平无明显改变。结论砷能诱导人正常膀胱上皮细胞VEGF表达增加,NAC能增加VEGF表达。

Antagonism of three kinds of antioxidants on arsenic-induced vascular endothelial growth factor expression in human urothelial cells

Objective To understand the antagonism of three kinds of antioxidants on arsenic-induced vascular endothelial growth factor (VEGF) expression in human urothelial cells.Methods SV-HUC-1 cells were treated with NaAsO2 at the doses of 0,0.5,1,2,4,8 and 10 μmol/L respectively for 24 h,the relative expression of VEGF was analyzed by using RT-PCR with β-actin as a loading control.Then SV-HUC-1 cells were treated with NaAsO2 at the doses of 4 and 10 μmol/L for 4,12,24,48,72 h,VEGF mRNA expression was analyzed by using RT-PCR.SV-HUC-1 cells were pretreated with DMSO and antioxidants (MEL,NAC,VC),the protein levels of VEGF were measured by Western blotting.Results VEGF mRNA expressions of NaAsO2 exposure groups increased with a significant dose-dependent manner,and showed significantly difference in 10 μmol/L NaAsO2 exposure group compared with that of control group (P＜0.05).The expressions of VEGF mRNA in SV-HUC-1 cells treated with 4 μmol/L NaAsO2 for 12,24,48 h and 10 μmol/L NaAsO2 for 24 and 48 h increased significantly compared with those of control group(P＜0.05).VEGF protein level increased in 4 μmol/L NaAsO2+DMSO group compared with those of control group(P＜0.05).Compared with NaAsO2+DMSO group,VEGF protein level increased in NAC+4 μ mol/L NaAsO2 group (P＜0.05),antioxidants (MEL and VC) decreased the protein expression of VEGF,but the difference was not statistically significant (P＞O.05).Conclusion Arsenic can induce VEGF expression in SV-HUC-1 cells,antioxidants NAC may increase the expression of VEGF protein.