| 摘要: |
| 目的 探讨JAK2抑制剂Ruxolitinib对人红白血病HEL细胞增殖、凋亡作用的机制。 方法 用不同浓度(0、1、5、10、50、100、500 nmol/L)的Ruxolitinib处理HEL细胞,其中0 nmol/L为对照组。CCK-8法检测细胞活力;Hoechst33342荧光染色检测细胞凋亡;流式细胞术检测细胞周期;罗丹明123检测线粒体膜电位变化;试剂盒检测Caspase-3/7活性;RT-PCR检测JAK2 mRNA水平;蛋白质印迹法检测p-JAK2、p-ERK、Bcl-2、Bim蛋白表达。 结果 不同浓度Ruxolitinib作用HEL细胞48 h后,细胞活力分别为(97.0±4.4)%、(92.0±3.9)%、(88.0±3.7)%、(81.0±3.1)%、(64.0±2.9)%、(38.0±2.2)%;Hoechst33342凋亡细胞染色显示100 nmol/L Ruxolitinib处理细胞48 h后,亮蓝色凋亡细胞[(49.21±1.80)%]较对照组[(10.02±1.40)%]增多(P<0.05);流式细胞术结果显示100 nmol/L Ruxolitinib作用细胞48 h后G0/G1期细胞比率[(73.1±3.6)%]高于对照组[(45.2±3.0)%];1~500 nmol/L Ruxolitinib作用12、24 h后,HEL细胞线粒体膜电位降低,Caspase-3/7活性增强;RT-PCR结果显示,不同浓度Ruxolitinib处理HEL细胞48 h后JAK2 mRNA表达呈剂量依赖性减低;蛋白质印迹检测结果显示,实验组细胞p-JAK2、p-ERK、Bcl-2蛋白表达较对照组降低(均P<0.01),Bim蛋白表达增加(P<0.01)。 结论 Ruxolitinib可能通过抑制JAK2及ERK激酶途径诱导HEL细胞凋亡。 |
| 关键词: 骨髓增殖性肿瘤 鲁索替尼 ERK Bcl-2 Bim |
| DOI：10.16781/j.0258-879x.2016.01.0052 |
| 投稿时间：2015-06-15修订日期：2015-08-03 |
| 基金项目:河北省科学技术研究与发展计划(08966107D), 2012年保定市科学技术研究与发展指导计划(12ZF105). |
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| Effect of Ruxolitinib on proliferation and apoptosis in human erythroleukemia leukemia cells |
| XU Qian1,2,LIU Gui-min1,2,GU Lei1,LIANG Wen-tong*1,CHENG Zhi-yong1* |
(1. Department of Hematology, The First Hospital of Baoding, Baoding 071000, Hebei, China;
2. Graduate School, Chengde Medical College, Chengde 067000, Hebei, China
*Corresponding authors.) |
| Abstract: |
| Objective To investigate the mechanism by which JAK2 inhibitor Ruxolitinib affecting the proliferation and apoptosis of human erythroleukemia leukemia(HEL) cells. Methods The HEL cells were treated with Ruxolitinib at different concentrations (1, 5, 10, 50, 100, and 500 nmol/L). Then the cell viability was detected by CCK-8 assay; the cell apoptosis was detected by Hochest staining method, the cell cycle was detected by flow cytometry, the mitochondrial membrane potential was assessed by rhodamine 123 with flow cytometry, and the Cysteine aspartic acid specific protease (Caspase)-3/7 protein activities were tested by kits. Moreover, the expression of JAK2 mRNA was measured by RT-PCR and the protein expressions of p-JAK2,p-ERK,Bcl-2 and Bim were observed by Western blotting analysis. Results After treated with different concentrations of Ruxolitinib at 1 nmol/L, 5 nmol/L,10 nmol/L, 50 nmol/L,100 nmol/L, and 500 nmol/L for 48 h, the HEL cell viabilities were (97.0±4.4)%,(92.0±3.9)%,(88.0±3.7)%,(81.0±3.1)%,(64.0±2.9)%,and (38.0±2.2)%, respectively. The ratio of high blue cells was significantly increased after treatment with 100 nmol/L Ruxolitinib for 48 h compared with the control group ([49.21±1.80]% vs [10.02±1.40]%, P<0.05). Flow cytometry showed G0/G1 phase cell ratio was higher after HEL cells exposed to 100nmol/L Ruxolitinib for 48 h compared with the control group ([73.1±3.6]% vs [45.2±3.0]%). The mitochondrial membrane potential was significantly decreased and Caspase-3/7 activity was significantly enhanced by treatment with Ruxolitinib at different concentrations (1-500 nmol/L) for 12 h and 24 h. RT-PCR showed that Ruxolitinib decreased JAK2 mRNA expression in a concentration-dependent manner in HEL cells after 48 h treatment.Western blotting analysis showed that the protein expressions of p-JAK2,p-ERK and Bcl-2 were significantly lower and Bim protein was significantly higher than those of control group(P<0.01). Conclusion Ruxolitinib may induce HEL cell apoptosis via JAK2 and ERK 1/2 pathways. |
| Key words: myeloproliferative neoplasms Ruxolitinib ERK Bcl-2 Bim |
