辛伐他汀诱导K562细胞凋亡过程中细胞内钙离子浓度、Calbindin-D28K和calpain基因的变化

Changes of expression levels of Ca^(2+),D28K(Calbindin-D28K) and calpain in K562 cell in the process of apoptosis induced by simvastatin

目的:观察辛伐他汀诱导K562细胞凋亡时细胞内Ca2+浓度、Ca2+调节蛋白(Calbindin-D28K,D28K)和Ca2+激活蛋白(calpain)基因变化,以探讨K562细胞凋亡机制。方法:常规培养细胞24 h,20μmol/L辛伐他汀处理K562细胞48 h;流式细胞技术检测细胞凋亡率和Ca2+浓度,PCR技术检测D28K和calpain基因。结果:20μmol/L辛伐他汀作用K562细胞24、48、72 h凋亡率改变分别为(6.10±0.35)%、(14.15±0.42)%、(30.70±0.65)%,随药物作用时间延长细胞凋亡率增加;辛伐他汀作用K562细胞12、24、48、72 h,游离钙荧光强度变化为52.93±2.85、19.63±1.56、13.81±1.05、7.84±0.98,各处理组荧光强度与对照组相比升高(P<0.05);与相同时间对照组比较,calpain和D28K基因分别在辛伐他汀作用K562细胞24 h和48 h后上调(P<0.05)。结论:辛伐他汀诱导K562细胞凋亡时细胞内Ca2+浓度显著升高,细胞内Ca2+浓度调节蛋白D28K和calpain基因上调,这些改变可能是辛伐他汀诱导K562细胞凋亡的机制之一。

Objective： To observe the changes of Ca2＋ level and calbindin-D28K and calpain gene,and investigate the mechanism in the process of simvastatin-induced apoptosis of K562 cells.Methods： K562 cells cultivated 24h were exposed in different concentrations of 20 μmol/L simvastatin.Flow cytometry was performed to confirm cell apoptotic ratio and analyze Ca2＋ level.The gene expression of D28K and calpain were analyzed by RT-PCR（reverse transcription-PCR）.Results： K562 cells could be induced to apoptosis after 20 μmol/L simvastatin treatment for 24 h,48 h and 72 h,and the apoptotic ratios were 6.1%±0.35%,14.15%±0.42%,and 30.70%±0.65% respectively by flow cytometric assay（Annexin V/PI double staining）.There was a significant increase in the drug-treated groups compared with the control groups after 24 h.The Ca2＋ level was detected by Fluo-3AM and was markedly increased in treated groups compared with the control groups at different time points（P0.05）.The mRNA of D28K and calpain expression levels in drug-treated group were up-regulated.Conclusion： K562 cells could be induced to apoptosis by simvastatin.The free of Ca2＋ may take part in the apoptosis in early period.The potential mechanism of the K562 cell apoptosis might be related tothe up-regulated calbindin-D28k and calpain gene.