Xu Guo-qiang, Pu Ze-yan, Liu Hua, Yang Yong-chang, Huang Wen-fang
Clinical Laboratory Department, Sichuan Academy Medical Sciences and Sichuan Provincial People's Hospital, Chengdu 610072, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2008 Nov;39(6):900-4.
To explore the apoptotic effect of simvastatin on K562 cells through Caspase-12 activation.
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 ([ca2+]i) 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.
Typical morphological changes of K562 apoptosis cells were observed post 72 hours treated with 10, 20, 30 micromol/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 micromol/L simvastatin, which represented the increase of [ca2+]i 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.
Caspase-12 is a important pathway of apoptosis in cells and participates simvastatin-induced apoptosis in K562 cells.
通过激活半胱天冬酶 - 12探讨辛伐他汀对K562细胞的凋亡作用。
采用Hoechst33258荧光染色在荧光显微镜下观察凋亡细胞的形态变化；用膜联蛋白V - FITC/PI双染法通过流式细胞术检测细胞凋亡率；用激光扫描共聚焦显微镜（LSCM）测量细胞内钙浓度（[ca2+]i）；用逆转录 - 聚合酶链反应（RT - PCR）检测葡萄糖调节蛋白78（GRP78）和钙蛋白酶基因mRNA的表达水平；用蛋白质免疫印迹法评估半胱天冬酶 - 3、-6、-7、-9、-12和GRP78蛋白的表达水平。
用10、20、30 μmol/L辛伐他汀处理72小时后，观察到K562凋亡细胞典型的形态变化。K562细胞的凋亡率分别为（12.41±0.32）%、（19.08±0.26）%和（23.41±0.36）%。用10、20、30 μmol/L辛伐他汀处理的K562细胞荧光强度分别为43±2.9、54±2.7和64±2.6，代表[ca2+]i升高。GRP78和钙蛋白酶基因mRNA的表达水平上调。蛋白质免疫印迹检测显示处理后的细胞中半胱天冬酶 - 3、-6、-7、-9、-12被切割激活以及GRP78表达上调。
半胱天冬酶 - 12是细胞凋亡的重要途径，参与辛伐他汀诱导的K562细胞凋亡。
