硒酸软骨多糖对K562细胞线粒体膜电位及凋亡的影响

Effects of Selenic Acid Cartilage Polysaccharide on Mitochondrial Membrane Potential and Cell Apoptosis of K562 Cells

本文研究了硒酸软骨多糖(CA-Se)对K562人慢性髓源白血病细胞线粒体膜电位的影响,及对K562细胞的凋亡诱导作用。采用MTT比色法测定CA-Se对K562细胞的增殖抑制作用;Hoechst 33342/PI染色后于显微镜下观察细胞的形态变化;罗丹明-123染色后用流式细胞仪检测细胞线粒体膜电位的变化;采用免疫荧光法检测caspase-9蛋白表达水平,并用RT-PCR检测其基因的表达。结果表明:CA-Se可显著抑制K562细胞的增殖,且具有浓度依赖性和时间依赖性;经CA-Se作用后,细胞形态发生明显改变,细胞体积变小,细胞膜变得不规则,染色质固缩;对照组与CA-Se处理组的线粒体膜电位分别为96.10%和78.07%,膜电位下降;CA-Se处理后,K562细胞中caspase-9蛋白表达显著增加,其基因表达水平显著上升。说明硒酸软骨多糖可通过降低细胞线粒体膜电位,上调凋亡起始酶caspase-9的基因表达,激活caspase-9蛋白来诱导K562细胞凋亡。

The effect of selenic acid cartilage polysaccharide(CA-Se) on the mitochondrial membrane potential of K562 human chronic myeloid leukemia cells and on the induction of apoptosis of K562 cells were studied. The inhibitory effect of CA-Se on the proliferation of K562 cells was studied using the MTT(3,-(4,5-dimethylthiazol-2-y))-2,5-diphenyl tetrazolium bromide) colorimetric assay, while the changes in the morphological characteristics after Hoechst 33342/PI staining were observed under a microscope. The changes in the mitochondrial membrane potential(ΔΨm) after rhodamine-123 staining were assessed by flow cytometry(FCM), and the protein and gene expressions of caspase-9 were analyzed by immunofluorescence and real-time polymerase chain reaction(RT-PCR), respectively. The results showed that the proliferation of K562 cells was significantly inhibited by CA-Se in a dose- and time-dependent manner. After CA-Se treatment, the morphological characteristics of cells were dramatically changed, and a reduction in cell volume, irregularities in cell membranes, and chromatin condensation were observed. The ΔΨm values reduced after CA-Se treatment from 96.10%(the control) to 78.07%(CA-Se treatment). The protein and gene expressions of caspase-9 both increased significantly. All the results indicated that CA-Se can induce apoptosis of K562 cells by reducing the ΔΨm, upregulating the expression of the caspase-9(initiator caspase) gene, and activating caspase-9 protein.