锰对HUVEC-304细胞生长及p53、p21^(WAF1/CIP1)蛋白表达的影响

Effects of manganese on the growth of HUVEC-304 cells and the expressions of p53,p21^(WAF1/CIP1) proteins

目的探讨锰对人脐静脉内皮细胞系HUVEC-304细胞生长及p53、p21WAF1/CIP1蛋白表达的影响。方法取指数生长期HUVEC-304细胞,以100、200、400、800μmol/L MnCl2,分别作用24、48、72 h后,以四甲基偶氮唑蓝(MTT)比色实验检测细胞的存活力,筛选锰对细胞的毒性剂量。流式细胞仪(FCM)检测细胞凋亡,Western blot方法检测p53、p21WAF1/CIP1蛋白的表达。结果100、200、400、800μmol/L MnCl2作用24、48、72 h均对HUVEC-304细胞有显著的抑制作用(P<0.05),且呈剂量-时间效应关系。流式细胞仪检测结果表明锰能诱导HUVEC-304细胞凋亡(P<0.01),Western blot结果显示,随锰浓度的增高,p53蛋白的表达下降(P<0.05);而p21WAF1/CIP1蛋白表达上升,差异有统计学意义(P<0.05),且呈剂量依赖性。结论锰可抑制HUVEC-304细胞的增殖,诱导其发生凋亡,p53蛋白的表达减少及p21WAF1/CIP1蛋白的表达增加是其发生凋亡的机制之一。

Objective To study the effects of manganese on the growth of HUVEC-304 cells and the expressions of p53 and p21^WAF1/CIP1 proteins. Method HUVEC-304 cells in exponential phase of growth were incubated in culture media with 100, 200, 400, 800 μmol/L manganese （MnC12） for 24, 48 and 72 h respectively; MTT colorimetry test was performed to examine the growth and survival of HUVEC-304 cells, flow cytometry （FCM） was used tn monitor cell apoptosis, and Western blot was used to measured the expression of p53 and p21^WAF1/CIP1 proteins. Result MTr measuring results revealed that manganese chloride （from 100 to 800 μmol/L） might suppress the proliferation of HUVEC-304 cells in dose and time-dependent manner （ P 〈 0.05） ; FCM results showed that manganese might induce the HUVEC-304 cells apoptosis. Western blot test found that manganese might down-regulate the expression of p53 proteins and up-regulate the express the p21^WAF1/CIP1 proteins, which were dose-dependent （P 〈 0.05）. Conclusion It was shown that manganese has obvious inhibition effect on the proliferation of HUVEC-304 cells and induee their apoptosis by down-regulate the expression of p53 proteins and up-regulate the expression of p21^WAF1/CIP1 proteins, which probably is one of the important mechanisms of manganese toxicity on HUVEC-304 cells.