辛伐他汀通过Caspase-12活化途径诱导K562细胞凋亡的实验研究

Experimental Study of Simvastatin Induced Apoptosis of K562 Cells by Caspase-12 Activation

目的探讨Caspase-12在辛伐他汀诱导K562细胞凋亡中的作用。方法在K562细胞培养液中分别加入毒胡萝卜素(阳性对照组)和辛伐他汀10、20、30μmol/L(辛伐他汀组)培养72 h,采用荧光显微镜观察凋亡细胞的形态变化,Annexin-FITC/PI双染法检测细胞凋亡率,激光扫描共聚焦显微镜检测细胞内Ca2+浓度,RT-PCR检测GRP78、Calpain基因mRNA表达水平,Western blot检测GRP78、Caspase-3,-6,-7,-9,-12蛋白水平。结果10、20、30μmol/L辛伐他汀作用K562细胞72 h后,细胞出现典型的凋亡形态,凋亡率分别为12.41%±0.32%、19.08±0.26%、23.41±0.36%;细胞内Ca2+浓度增加,荧光强度分别为43±2.9、54±2.7、64±2.6;GRP78、Calpain基因mRNA表达上调;Western blot检测显示:Caspase-3,-6,-7,-9,-12剪切活化,GRP78表达增强。结论Caspase-12作为细胞凋亡的重要途径参与了辛伐他汀诱导K562细胞的凋亡。

Objective To explore the apoptotic effect of simvastatin on K562 cells through Caspase-12 activation. Methods Morphological changes of apoptotic cells were observed by Hoechst33258 fluorescent staining under fluorescent microscope; Apoptosis rate of cells was determined with annexin V-FITC/PI double staining by flow cytometry; Intracellular calcium concentration （[ca^2＋]） was measured by Laser Scanning Confocal Microscope（LSCM） ; The expression levels of GRP78 and Calpain gene mRNA were determined by RT- PCR; The expression levels of Caspase-3,-6,-7,-9,-12 and GRP78 proteins were evaluated by Western blot. Results Typical morphological changes of K562 apoptosis cells were observed post 72 hours treated with 10, 20, 30 μmol/L simvastatin. The apoptotic rates of K562 cells were （12.41±0. 32）%, （19.08±0. 26）% and （23.41± 0.36）%, respectively. The fluorescent intensities were 43±2.9, 54±2.7 and 64±2.6, respectively in K562 cells treated with 10, 20, 30 μmol/L simvastatin, which represented the increase of [ca^2＋]. The expression levels of GRP78 and Calpain gene mRNA were up-regulated. And the cleavage and activation of Caspase-3 ,-6 ,-7 ,-9 ,-12 and upregulation of GRP78 expression were demonstrated by Western blot detection for the treated cells. Conclusion Caspase-12 is a important pathway of apoptosis in cells and participates simvastatin-induced apoptosis in K562 cells.