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目的 探讨氨基葡萄糖硫酸盐(GS)对慢性髓系白血病K 5 62细胞凋亡诱导作用和分子机理。方法 采用台盼蓝拒染法测定细胞生长曲线来观察GS对K5 62细胞的生长抑制作用;取对数生长期K 5 62细胞,在0 .5 ,1.0 ,5 .0mmol/LGS不同浓度作用48h后,利用细胞计数、电镜、吖啶橙/溴化乙啶(AO/EB)染色、DNA梯状电泳、流式细胞仪等方法来观察GS对K 5 62细胞的抑制效应和促凋亡作用。并用Westernblot检测活化的caspase 3和bcl- 2基因的表达。结果 0 .5 ,1.0 ,5 .0mmol/LGS对K 5 62细胞的生长有抑制作用,并呈时间和剂量依赖性(P <0 .0 1)。采用光镜、电镜和AO /EB染色法发现5mmol/LGS作用K 5 62细胞后,出现典型的凋亡形态学改变。AnnexinV染色后流式细胞仪能检测到早期凋亡细胞,细胞周期显示:G1期细胞比例增高,S期比例减低,并有凋亡峰。有明显的梯状DNA。同时GS诱导K 5 62细胞凋亡后出现活化的Caspase 3 ,而Bcl- 2蛋白表达减弱,甚至消失。结论 GS能诱导白血病K- 5 62细胞凋亡,该过程伴有Caspase- 3的活化和Bcl- 2蛋白表达降低。
Objective To investigate the apoptosis-inducing effect and molecular mechanism of glucosamine sulfate (GS) on chronic myeloid leukemia K 5 62 cells. Methods The growth curve of GS on K562 cells was observed by trypan blue dye exclusion method. K 5 62 cells in logarithmic growth phase were treated with different concentrations of 0.5, 1.0, 5.0 mmol/LGS for 48 h. Then, the inhibitory and proapoptotic effects of GS on K 5 62 cells were observed by cell counting, electron microscopy, acridine orange/ethidium bromide (AO/EB) staining, DNA ladder electrophoresis, and flow cytometry. . The expression of activated caspase 3 and bcl-2 gene was detected by Western blot. Results 0.5, 1.0, and 5.0 mmol / LGS inhibited the growth of K562 cells in a time and dose dependent manner (P <0.01). Using light microscopy, electron microscopy and AO/EB staining, 5 mmol/LGS K 5 62 cells were found to undergo typical apoptotic morphological changes. After Annexin V staining, early apoptotic cells were detected by flow cytometry. The cell cycle showed that the proportion of G1 phase cells increased, the proportion of S phase decreased, and there was a peak of apoptosis. There is obvious laddering DNA. At the same time, GS induces the activation of Caspase 3 after apoptosis of K 5 62 cells, while Bcl-2 protein expression is weakened or even disappeared. Conclusion GS can induce apoptosis of leukemia K-562 cells, which is accompanied by activation of Caspase-3 and decreased expression of Bcl-2 protein.