镉诱导293细胞凋亡与Caspase-3和p53表达关系

The apoptosis of 293 cell induced by cadmium associated with the expression of Caspase-3 and p53

目的 研究镉致细胞凋亡过程中Coapase-3和p53表达的变化，探讨Caspase-3和p53基因在镉致细胞调亡中的作用。方法离体培养的转化人胚肾293细胞以40μmol／L氯化镉分别处理0—24h，N-乙酰半胱氨酸（NAC）和Caspase-3特异性三肽抑制剂（Z—DEVD-fmk）预处理组分别在镉处理前以5mmol／LNAC和10μmol／L Z-DEVD-fmk预处理24h和1h，逆转录-聚合酶链反应（RT-PCP,）和Westem blot法测Coapase-3和茚3水平，流式细胞Ancxin-V-PI双粢法检测细胞凋亡率。结果40μmol／L镉处理293细胞，6—24 h Coapoae-3基因mRNA水平显著增高，0—24h内p53基因mRNA无显著变化。40μmol／L镉处理293细胞，6—24h内Caspase-3相对分子质量20000的活性亚单位条带显著增强，与镉处理24h组比较，NAC顸处理组Caspase-3相对分子质量20000的活性亚单位条带显著减弱（P〈0．01），Z-DEVD—fmk预处理组无明显变化（P〉0．05）；40μmol／L镉处理293细胞，郦蛋白在6—24h表达增强（P〈0．01），与单纯镉处理24h组比较，NAC和z—DEVD-fmk预处理组筇3蛋白表达无显著变化（P〉0．05）。结论Caspase-3基因在镉诱导细胞调亡过程中起着重要的作用。

Objective To study the relationship between the apoptosis of 293 ceU induced by cadmium and the expression of Caspaze-3 and p.53. Methods Transformed human embryonic kidney 293 cell line were incubated with 40 μmol/L CdCh for 0 - 24 hours. The ceUs in the groups of NAC and Z-DEVD-fmk pretreatment were treated with 5.0 μmol/L NAC and 0. 1 μmol/L Z-DEVD-fmk before incubated with 40 μmol/L CdCh. The expressions of Caspase-3 and p53 gene in 293 cell after treated with cadmium were determined by the methods of reverse transcription polymerase chain reaction （RT-PCR） and Western-blot analysis. And the occurrence of apoptosis was determined by flowcytometry. Results RT-PCR analysis revealed that incubated ceils with 40 μmol/L cadmium 6 - 24 hours, the expression of Caspase-3 mRNA were increased obviously when compared with the controls. The pretreatment of NAC and Z-DEVD-fmk increased the expression of Caspase-3 mRNA compared with the cells with 40 μmol/L cadmium only. The expression of p53 mRNA had no changes between 0 - 24 hours incubated cells with 40 μmol/L cadmium. Western-blot analysis revealed that Caspase-3 20 000 u and p53 protein were increased 6 hours after cells incubated with 40 μmol/L cadmium. Pretreatment with NAC decreased the Caspase-3 20 000 u while pretreatmcnt with Z-DEVD-fmk had no effects on it. Both NAC and Z-DEVD-fmk had no effects on the expression of p53 protein. Conclusion It was suggested that Caspase-3 could play a key role in cadmium-induced cell apoptosis.