上调CaMKⅡ抑制蛋白表达对HL-60细胞增殖的抑制作用及机制研究

摘要

Objective To investigate the inhibitory effects of CaMK Ⅱ N on acute myeloid leukemia cell line HL-60 to explore a novel therapeutic target of leukemia.Methods Human CaMK Ⅱ N gene expression vector pcDNA3.1/hCaMK Ⅱ N or empty vector pcDNA3.1/myc-His (-) B was transfected into HL-60 cells by Lipofectamine 2000.Human CaMK Ⅱ N proteins of transfected cells were detected by Western blot.Cell proliferation affected by human CaMK Ⅱ N was determined by MTT.Colony-forming assay was performed by soft agar growth system.The cells transfected with CaMK Ⅱ N were stained with Hoechst 33342 to detect the apoptotic proportion under fluorescence microscopy.Cell cycle was analyzed by flow cytometry.Results Human CaMK Ⅱ N was stably transfected into HL-60 cells,and overexpression of human CaMK Ⅱ N inhibited the proliferation ofHL-60/CaMK Ⅱ N cells compared to HL-60/mock cells and HL-60 cells [(0.44+0.03)vs(0.94±0.05)vs(0.94±0.04)P＜0.01].The colony formation of HL-60/CaMK Ⅱ N was also markedly smaller [(21.00±3.05)/500)] than that of mock-transfected [(111.00±4.58)/500)] and control cells [(119.00 ± 6.09)/500)] (P＜0.01).After 72 hrs-culture,the apoptotic proportion in cells transfected with CaMK Ⅱ N was obviously higher than of cells transfected with mock DNA or control [(22.49±2.15)％ vs(7.17±0.72)％ vs(6.40±0.55)％,P＜0.01].Up to(82.97±2.90)％ human CaMK Ⅱ N/HL-60 cells were arrested at G0/G1 phase,which was more than mock-transfected [(40.53±2.38)％] and control cells [(41.63 ± 2.27)％] (P＜0.05).Human CaMK Ⅱ N could down-regulate expression of Bcl-2 in transfected cells.Conclusion CaMK ⅡN up-regulation could inhibit proliferation and induce apoptosis of human acute myeloid leukemia cell HL-60.%目的 观察上调钙/钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)抑制蛋白(CaMKⅡN)基因表达对急性髓系白血病(AML)细胞增殖的影响及机制.方法 以白血病细胞系HL-60细胞为研究对象,采用Lipofectamine 2000脂质体法将CaMKⅡN基因真核表达载体pcDNA3.1/CaMKⅡN和空载体pcDNA3.1/myc-His(-)B分别转染细胞;Western blot法检测转染后的HL-60细胞CaMKⅡN基因表达;MTT法检测转染CaMKⅡIN基因的细胞增殖情况;半固体培养法分析转基因HL-60细胞集落形成情况;Hoechst33342荧光染色观察细胞凋亡;流式细胞术检测细胞周期分布.结果 人CaMKⅡN基因稳定转染HL-60细胞.过表达CaMKⅡN基因的HL-60细胞与空载体转染组及空白对照组相比,增殖受抑[(0.44±0.03)对(0.94±0.05)和(0.94±0.04),P＜0.01],细胞集落形成能力减低[(21.00±3.05)/500细胞对(111.00±4.58)/500细胞和(119.00±6.09)/500细胞,P＜0.01],转染72 h细胞凋亡率明显增高[(22.49±2.15)％对(7.17±0.72)％和(6.40±0.55)％,P＜0.01].过表达CaMKⅡN的HL-60细胞阻滞于G0/G1期[(82.97±2.90)％],明显高于空转组[(40.53+2.38)％]和空白对照组[(41.63+2.27)％](P＜0.05);转染CaMKⅡN基因的HL-60细胞Bcl-2表达水平明显降低.结论 上调人CaMKⅡN基因表达可有效抑制HL-60细胞增殖,诱导其凋亡.