报告基因方法监测重金属汞及其化合物的早期毒性

目的：基于热休克信号响应和分泌型碱性磷酸酶（SEAP）报告基因,建立HSE-SEAP-HeLa细胞模型,预测重金属（汞及其化合物）的早期毒性。方法：将pHSE-SEAP质粒转染到人子宫颈癌HeLa细胞中,建立瞬时HSE-SEAP响应模型。热休克（42 ℃,1 h）或重金属化合物（CdCl₂,5 μmol·L^-1,4 h）处理后,在完全DMEM培养基中恢复48 h后,检测细胞上清液中的分泌型碱性磷酸酶的含量（表示该状态下细胞中的热休克蛋白表达量）,同时用MTT方法检测对应浓度下的细胞活性,以验证模型的有效性和重复性。并用亚致死浓度（由MTT实验确定）的无机和矿物汞（HgCl₂,HgS和朱砂）和柳硫汞钠处理细胞,检测诱导的细胞热休克响应。结果：热休克和CdCl₂作用HSE-SEAP-HeLa细胞模型后,细胞上清液中的分泌型碱性磷酸酶的含量显著升高；热休克蛋白表达早于Cd²⁺对细胞活性的降低。4种汞化合物在亚致死浓度均诱导细胞内热休克蛋白的变化,但4种化合物的作用时间和浓度效应不同,显示不同类型的汞化合物早期毒性存在差异。结论：HSE-SEAP-HeLa细胞模型可有效检测重金属诱导的热休克应激作用,并具有良好的重复性,可应用于重金属有关的早期毒性预测或药物毒性评估。

Monitoring early toxicity of heavy metals including Hg using a HSE-SEAP reporter gene

OBJECTIVE: To develop a cellular assay based on heat shock signal pathway and secreted alkaline phosphatase (SEAP) reporter gene for investigating/predicting the early toxicity of heavy metals on HeLa cells in Chinese traditional medicine (TCM). METHOD: The pHSE-SEAP plasmid was transfected into HeLa cells to build a HSE-SEAP-HeLa cell model. For validation of the model, the transfected cells were treated by either heating at 42 degrees C for 1 h or incubated with 5 mol x L(-1) CdCl2 for 4 h. Then the cells were covered in complete DMEM culture medium for 48 h and the activity of SEAP (reflecting the cellular level of heat shock protein) in cultural supernatants was measured; meanwhile, cell viability was determined by MTT assays. In addition, the cells were treated by four mercury compounds, HgCl2, merthilate sodium, HgS and cinnabar at the sub-lethal concentrations (determined by MTT assays). Then the heat shock response was detected likewise. RESULT: Significant level of secreted alkaline phosphatase (SEAP) was found in pHSE-SEAP transfected HeLa cells treated either by heating (42 degrees C) or incubating with CdCl2. The heat shock protein was induced by CdCl2 before decrease of cell viability was observed. All four mercury compounds induced heat shock response in both time and concentration-dependant manner. However, there were big differences among the mercury compounds, suggesting potential differences for early-stage toxicity in vivo. CONCLUSION: The pHSE-SEAP transfected HeLa cells respond effectively to heat shock and metal stresses, and therefore provide a practical and repeatable assay for investigating/predicting the early toxicity of heavy metals and mineral-containing drugs in TCM.