摘要
目的探讨二甲基甲酰胺（N，N—dimethylformamide，DMF）对人肝细胞（HL-7702）钙稳态与钙蛋白酶（calpain）I、Ⅱ基因表达的影响。方法分别以10、25、50、100、200mmol／LDMF对人肝细胞染毒，空白对照组加等体积的DMEM，采用钙离子荧光探针（Fluo-3，AM）技术动态监测人肝细胞内Ca^2＋浓度的变化。以10、25、50、100、150、200mmol／L剂量染毒24h后，倒置相差显微镜观察细胞形态，噻唑蓝法观察各组细胞存活率。以10、25、50、100、150mmol／L剂量DMF染毒24h，空白对照组加入等体积的DMEM，通过实时定量PCR检测各组calpainI、Ⅱ的mRNA表达水平。结果肝细胞经不同浓度DMF处理24h后，各组的细胞存活率差异有统计学意义（P〈0．01），50、100、150、200mmol／L剂量组的细胞存活率明显低于对照组，差异有统计学意义（R0．05）。倒置相差显微镜观察发现，随着DMF染毒剂量的增加，肝细胞逐渐失去原来的形状，出现肿胀、回缩等损伤现象，150mmol／L染毒组细胞甚至出现变圆、漂浮等现象。随着DMF染毒剂量增加，细胞内Fluo-3的荧光强度升高，差异有统计学意义（P〈0．05），存在剂量一反应关系。各剂量组calpainI和calpain11mRNA相对表达量的差异有统计学意义（P〈0．01），且随着染毒剂量的增加而升高，但DMF浓度达150mmol／L时，却对calpainImRNA的表达没有促进作用。结论DMF引起的肝损伤与细胞内的钙稳态失调及calpainmRNA水平增加有关。
Objective To investigate the effect of N,N-dimethylformamide （DMF） on calcium home- ostasis and calpain I and II gene expression in human hepatocytes （HL-7702）. Methods HL-7702 cells were exposed to different concentrations of DMF （10, 25, 50, 100, or 200 mmol/L）; other HL-7702 cells, which were used as a control group, were exposed to the equal volume of DMEM; the intracellular Ca2＋ concentration was monitored using the calcium fluorescent probe （fluo-3/AM）. After 24-h exposure to DMF （ 10, 25, 50, 100, 150, or 200 mmol/L）, the morphology of hepatocytes was observed under an inverted phase contrast microscope, and the cell viability was measured by MTT assay. After 24-h exposure to DMF （ 10, 25, 50, 100, or 150 mmol/L）, the mRNA expression levels of calpain I and II in hepatncytes were measured by real-time quantitative PCR. Results There were significant differences in cell viability among different exposure groups （P〈0.01）; the 50, 100, 150, and 200 mmol/L DMF exposure groups had a significantly lower cell viability than the control group （P〈0.05）. Under the inverted phase contrast microscope, HL-7702 cells gradually lost the original shape, with swelling and shrinking, as the dose of DMF increased, and those treated with 150 mmol/L DMF even became round and floated. The fluorescence density of fluo-3 in hepatocytes increased as the dose of DMF rose, demon- strating a dose-response relationship, and there were significant differences among these exposure groups （P〈0.05）. There were significant differences in mRNA expression levels of calpain I and II among these exposure groups （P〈0.01）, and the expression increased as the dose of DMF rose; but DMF did not promote the mRNA expres- sion of calpain I at a concentration of 150 mmol/L. Conclusion DMF can cause damage to hepatocytes, which is related to intracellular calcium increase and calpain mRNA increase.
出处
《中华劳动卫生职业病杂志》
CAS
CSCD
北大核心
2013年第3期184-188,共5页
Chinese Journal of Industrial Hygiene and Occupational Diseases
基金
浙江省自然科学基金项目（Y207472）
浙江省医学支撑学科劳动卫生学（11-ZC02）
（郑晓亮、蒋兆强、朱丽瑾、肖芸、鞠莉、张敏在本实验中给予了大力支持和帮助志谢!）
