目的:研究丝/苏氨酸蛋白激酶Pim-1抑制剂SMI-4a对人类急性髓系白血病细胞株U937的生长抑制、促凋亡作用及其可能机制.方法:CCK-8法检测不同浓度SMI-4a作用不同时间对U937细胞的生长抑制率;Annexin V-PI及Hoechst 33342染色法检测SMI-4a作用前后细胞凋亡情况,集落形成实验检测SMI-4a对U937细胞集落形成能力的影响;Western blot法检测SMI-4a对U937细胞核及细胞浆内β-catenin表达变化及细胞内凋亡相关蛋白的表达变化;免疫荧光法检测β-catenin在细胞内的表达变化.结果:CCK-8结果显示SMI-4a可以抑制U937细胞的活力,并呈时间和剂量依赖性;Annexin V-PI及Hoechst 33342染色结果显示SMI-4a可以促进U937细胞凋亡;集落形成实验证实SMI-4a可以抑制U937细胞的集落形成能力;Western blot实验结果显示SMI-4a作用于U937细胞48 h后细胞浆内的β-catenin表达增加,细胞核内的β-catenin表达减少,细胞内促凋亡蛋白Bax和PARP表达增强,抑凋亡蛋白Bcl-2表达明显减弱;免疫荧光进一步验证了SMI-4a作用后的U937细胞核内的β-catenin表达量明显减少.结论:SMI-4a诱导U937细胞凋亡是通过上调促凋亡基因的表达、下调凋亡抑制基因的表达来实现的.%AIM:To study the growth-inhibiting and proapoptotic effects of Pim-1 kinase inhibitor SMI-4a on human acute myeloid leukemia cell line U937.METHODS:The effect of SMI-4a on U937 cell viability was measured by CCK-8 assay.The apoptotic rate was assessed by flow cytometry with Annexin V-PI staining and by fluorescence microscopy with Hoechst 33342 staining.Methylcellulose was used to assess colony formation ability of the cells.The expression of β-catenin in the cell cytosol and nucleus was detected by Western blot,and the expression of apoptosis-related proteins in the U937 cells was also examined.Intracellular distribution of β-catenin was detected by the method of immunofluorescence.RESULTS:SMI-4a inhibited the viability of U937 cells.Annexin V-PI staining showed that SMI-4a induced apoptosis in dose-and time-dependent manners.Hoechst 33342 staining also verified the apoptosis.SMI-4a significantly inhibited the colony formation capacity of the U937 cells.The results of Western blot demonstrated that SMI-4a upregulated the expression of PARP and Bax,downregulated the expression of Bcl-2 and change the distribution of β-catenin in intracellular compartment.Immunofluorescence observation found that SMI-4a decreased the expression level of β-catenin in the U937 cells.CONCLUSION:SMI-4a induces U937 cell apoptosis through regulating the expression of apoptosis-related genes.
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