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  

Inhibiting effect of CaMK Ⅱ N up-regulation on leukemia cells growth and its mechanism

作　　者：侯军[1] 施菊妹[1] 韩颖[1] 胡晓静[1] 孟秀琴[1] 

机构地区：[1]同济大学附属第十人民医院血液科,上海200072

出　　处：《中华血液学杂志》2013年第12期1006-1009,共4页Chinese Journal of Hematology

基　　金：上海市卫生局资助项目（2010081）

摘　　要：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Lipofectamine2000脂质体法将CaMK1IN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；Westernblot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72h细胞凋死率明显增高[（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。结论上涮人CaMKⅡN基因表达可有效抑制HL-60细胞增殖，诱导其凋亡。Objective To investigate the inhibitory effects of CaMK ⅡN 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 ⅡN was determined by MTT. Colony-forming assay was performed by soft agar growth system. The cells transfected with CaMK ⅡN were stained with Hoechst 33342 to detect the apoptotic proportion under fluorescence microscopy. Cell cycle was analyzed by flow cytometry. Results Human CaMK ⅡN was stably transfected into HL-60 cells, and overexpression of human CaMK 1/N inhibited the proliferation of HL-60/CaMK ⅡN cells compared to HL-60/mock cells and HL-60 cells [ （0.4±0.03）vs（0.94±0.O5）vs（0.94＋0.04）P〈0.011. The colony formation of HL-60/CaMK ⅡN was also markedly smaller [ （21.00±3.05）/500） 1 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〈0.011. Up to（82.97±52.90）% human CaMK Ⅱ N/HL-60 cells were arrested at G0/G1 phase, which was more than mock-transfected E （40.53±2.38）% ] and control cells [ （41.63±2.27）% 1 （P〈0.05）. Human CaMK ⅡN could down-regulate expression of Bcl-2 in transfected cells. Conclusion CaMK Ⅱ N upregulation could inhibit proliferation and induce apoptosis of human acute myeloid leukemia cell HL-60.

关 键 词：CaMKIIN 基因表达 HL-60细胞 基因 BCL-2 

分 类 号：R363[医药卫生—病理学]