p53和γ-H2AX作为乙醛引起DNA损伤早期生物标记物的实验研究

Changes of p53 and γ-H2AX of TK6 cells caused by acetaldehyde

目的：评价乙醛染毒p53野生型人类淋巴母细胞（TK6）后，细胞中DNA损伤标记物p53、γ-H2AX的表达变化，并与彗星试验中的DNA链断裂指标进行敏感性比较，探讨p53和γ-H2AX作为乙醛引起DNA损伤的早期生物标记物的可能。方法：乙醛在0.5～20 mmol/L的浓度范围分别染毒TK6细胞20 min、2和12 h后，采用高通量的流式细胞术检测肿瘤抑制蛋白total-p53、磷酸化p53（p-p53）以及磷酸化组蛋白（γ-H2AX）的细胞表达率和表达强度（平均荧光强度）；同时采用碱性彗星试验检测乙醛引起的DNA链断裂指标（细胞拖尾率、尾长、尾部DNA百分含量以及尾矩）的变化。结果：乙醛染毒TK6细胞12 h后，γ-H2AX的表达强度在15及20 mmol/L浓度下显著升高（P〈0.05），p-p53的细胞表达率在0.5～7.5 mmol/L浓度范围内呈现剂量依赖的升高趋势，total-p53的细胞表达率趋势与p-p53相似，但与阴性对照组相比，差异无统计学意义。彗星试验中，细胞拖尾率、尾长、尾部DNA百分含量以及尾矩在5.0～12.5 mmol/L浓度范围内均增加，并呈现剂量依赖性。染毒2 h后，与阴性对照组相比，total-p53和p-p53的细胞表达率在15和20 mmol/L浓度下显著升高（P均〈0.05），细胞拖尾率、尾部DNA百分含量以及尾矩在20 mmol/L浓度升高（P均〈0.05）。染毒20 min后，3种生物标志物均未见清晰的变化趋势，4种DNA链断裂指标均未见显著变化（P均＞0.05）。结论：乙醛染毒TK6细胞12 h可诱导p-p53细胞表达率升高、γ-H2AX细胞表达强度升高、total-p53细胞表达率轻微升高，以及细胞拖尾率、尾长、尾部DNA百分含量、尾矩的升高。p-p53比γ-H2AX和DNA链断裂指标在检测乙醛诱导的DNA损伤方面更加敏感。

OBJECTIVE： The aim of this study was to investigate the changes of biomarkers of DNA damage,total p53,phospho-p53 （p-p53） and phospho-H2AX （p-H2AX orγ-H2AX） following exposure of TK6 cells to acetaldehyde and to explore the roles and the sensitivity of p53,γ-H2AX in DNA damage detection by comparison in the indicators of DNA strand breaks （comet assay）. METHODS：TK6 cells were exposed to acetaldehyde with different concentrations of 0.5-20 mmol/L for 20 min,2 and 12 h. Flow cytometry was applied to investigate the changes of relevant biomarkers of DNA damage. Alkaline comet assay was used to detect the indicators of DNA strand breaks （percentage of cells with tails,tail length,percentage of tail DNA,tail moment）. RESULTS：After exposure to acetaldehyde for 12 h,a significant increase ofγ-H2AX expression was observed at acetaldehyde doses of 15 and 20 mmol/L. A significant dose-dependent increase of p-p53 expression rate was found at acetaldehyde dose between 0.5-7.5 mmol/L. In parallel,a similar trend of expression was observed in total p53,but no significant increase was shown. For the comet assay,significant dose-dependent increases of percentage of cells with tails,tail length,percentage of tail DNA,tail＆amp;nbsp;moment were obtained after 12 h exposure acetaldehyde dose between 5.0-12.5 mmol/L. 2 h exposure showed that both total p53 and p-p53 expression rates increased significantly at concentrations of 15 and 20 mmol/L of acetaldehyde,and significant increases of percentage of cells with tails,tail DNA,tail moment accurred at the concentration of 20 mmol/L acetaldehyde,but no response was observed in γ-H2AX. For 20 min exposure,no clear trend for all the three biomarkers and no significant change were shown with the indicators of DNA strand breaks. CONCLUSION：Acetaldehyde could induce increase of p-p53 expression rate,γ-H2AX expression amount,percentage of cells with tails,tail length,percentage of tail DNA,tail moment and total p53 expression rate to a less extent. The biomarker p-p53 may be more sensitive reflecting the DNA damage caused by acetaldehyde than γ-H2AX and indicators of DNA strand breaks.