CAJ | 학술논문

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、200 mmol/L DMF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Ca2+浓度的变化.以10、25、50、100、150、200 mmol/L剂量染毒24 h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、150 mmol/L剂量DMF染毒24 h,空白对照组加入等体积的DMEM,通过实时定量PCR检测各组calpain Ⅰ、Ⅱ的mRNA表达水平.结果肝细胞经不同浓度DMF处理24 h后,各组的细胞存活率差异有统计学意义(P＜0.01),50、100、150、200 mmol/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(P＜0.05).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,150 mmol/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-反应关系.各剂量组calpain Ⅰ和calpainⅡmRNA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150 mmol/L时,却对calpain Ⅰ mRNA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calpain mRNA水平增加有关.
목적 탐토이갑기갑선알(N,N-dimethylformamide,DMF)대인간세포(HL-7702)개은태여개단백매(calpain)Ⅰ、Ⅱ기인표체적영향.방법 분별이10、25、50、100、200 mmol/L DMF대인간세포염독,공백대조조가등체적적DMEM,채용개리자형광탐침(Fluo-3,AM)기술동태감측인간세포내Ca2+농도적변화.이10、25、50、100、150、200 mmol/L제량염독24 h후,도치상차현미경관찰세포형태,새서람법관찰각조세포존활솔.이10、25、50、100、150 mmol/L제량DMF염독24 h,공백대조조가입등체적적DMEM,통과실시정량PCR검측각조calpain Ⅰ、Ⅱ적mRNA표체수평.결과 간세포경불동농도DMF처리24 h후,각조적세포존활솔차이유통계학의의(P＜0.01),50、100、150、200 mmol/L제량조적세포존활솔명현저우대조조,차이유통계학의의(P＜0.05).도치상차현미경관찰발현,수착DMF염독제량적증가,간세포축점실거원래적형상,출현종창、회축등손상현상,150 mmol/L염독조세포심지출현변원、표부등현상.수착DMF염독제량증가,세포내Fluo-3적형광강도승고,차이유통계학의의(P＜0.05),존재제량-반응관계.각제량조calpain Ⅰ화calpainⅡmRNA상대표체량적차이유통계학의의(P＜0.01),차수착염독제량적증가이승고,단DMF농도체150 mmol/L시,각대calpain Ⅰ mRNA적표체몰유촉진작용.결론 DMF인기적간손상여세포내적개은태실조급calpain mRNA수평증가유관.
Objective To investigate the effect of N,N-dimethylformamide (DMF) on calcium homeostasis 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 hepatocytes 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,demonstrating 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 expression 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.