冬凌草甲素对白血病细胞增殖抑制及机制的研究

Anti-Leukemia Effect and Mechanism of Oridonin on Imatinib-Sensitive and Imatinib-Resistant K562 Cells

目的:探讨伊马替尼耐药的K562细胞(K562-R)的可能耐药机制及冬凌草甲素(oridonin,OR)对伊马替尼敏感的K562细胞(K562-S)和K562-R细胞的生长抑制作用及其可能机制。方法:Western blot法检测K562-S和K562-R细胞内p-Lyn的表达;采用改良的四甲基偶氮唑蓝(MTT)法检测OR对K562-S和K562-R细胞的增殖抑制作用,光学显微镜观察OR作用24 h后K562-S和K562-R细胞的形态变化,流式细胞术检测细胞凋亡率,Western blot法检测OR对K562-S和K562-R细胞内BCL-2的表达和蛋白激酶p-Lyn、Akt/mTOR信号通路的变化。结果:在K562-R细胞中,p-Lyn表达增高。OR对K562-S和K562-R细胞的生长具有抑制作用,且呈时间和浓度依赖性,OR作用72 h的半抑制浓度(IC50)值分别为4.23±1.30和4.97±2.23μmol/L。OR作用24 h后,K562-S和K562-R细胞破坏明显,部分细胞裂解成碎片。流式细胞仪检测发现,OR使K562-S和K562-R凋亡细胞明显增多。Western blot结果提示,OR使细胞内蛋白激酶p-Lyn表达下降,Akt/mTOR信号通路关键点蛋白磷酸化水平明显减低,凋亡抑制蛋白BCL-2表达下调。结论:p-Lyn高表达可能参与K562-R细胞的耐药。OR通过下调p-Lyn,抑制Akt/mTOR信号通路和下调BCL-2表达,促进细胞凋亡,抑制白血病细胞株K562-S和K562-R的生长。

Objective： To explore the molecular mechanism of resistance to imatinib in K562 cells（K562-R） and the anti-proliferative effect of oridonin（OR）,as well as its mechanism in imatinib-sensitive and imatinib-resistant K562 cells（K562-S and K562-R cells）. Methods： The expression of p-Lyn in K562-S and K562-R cells were detected by Western blot. The anti-proliferative effect of OR in K562-S and K562-R cells was assayed by MTT,the morphological changes were examined with microscope,the cell apoptosis was detected by flow cytometry,the expressions of BCL-2 and Akt/mTOR signaling pathway were detected by Western blot. Results： The over-expression of p-Lyn was detcected in K562-R cells,OR inhibited the proliferation of K562-S and K562-R cells and the value of IC50 was 4. 23 ± 1. 30,4. 97 ± 2. 23 μmol/L,respectively. The apoptotic rate was obviously enhanced after OR treatment for 24 h,compared with control group. OR down-regulated the expression of p-Lyn,mTOR signaling pathway and BCL-2 protein. Conclusion： Over-expression of pLyn may be involved in the mechanism of resistance to imatinib. OR can inhibit the proliferation of K562-S and K562-R cells through down-regulating p-Lyn,inhibiting mTOR signaling pathway and down-regulating expression of BCL-2 protein.