自噬调控对JAK2V617F阳性HEL细胞及真性红细胞增多症患者造血细胞增殖的影响  

The impact of autophagy on proliferation of HEL cells and hematopoietic cells of polycythemia vera patients with JAK2 V617F mutation

作　　者：董露[1] 申徐良[2] 魏武[2] 史文芝[2] 张国香[2] 曹文君[3] 李丹[3] 

机构地区：[1]山西医科大学第一临床医学院,太原030001 [2]长治医学院附属和平医院血液内科 [3]长治医学院附属和平医院中心实验室

出　　处：《中华血液学杂志》2015年第6期520-525,共6页Chinese Journal of Hematology

基　　金：山西省自然科学基金（2013011056-3）;山西省高校科技开发项目（20121013）;山西省卫生厅科研课题（20122008）

摘　　要：目的 通过检测JAK2 V617F阳性HEL细胞及真性红细胞增多症（PV）患者造血细胞自噬水平,探索自噬调控对HEL细胞及PV患者造血细胞增殖的影响.方法 应用流式细胞术、吖啶橙染色法、Western blot法检测JAK2 V617F阳性HEL细胞和12例PV患者造血细胞中LC3-Ⅱ蛋白的表达水平;通过雷帕霉素和3-甲基腺嘌呤（3-MA）分别诱导和抑制HEL细胞及3例PV患者骨髓细胞自噬水平,应用上述方法检测细胞中LC3-Ⅱ蛋白表达水平的改变,并采用CellTiter-Glo（R）发光法检测细胞增殖活力.结果 HEL细胞内LC3-Ⅱ蛋白的平均荧光强度（159 389±29 001）及自噬体水平明显高于JAK2 V617F阴性K562细胞（96 047±24 134）（P=0.044）,PV患者外周血中髓系细胞内LC3-Ⅱ蛋白平均荧光强度（92 842±4 250）明显高于健康志愿者（86 633±2 504）（P=0.001）;自噬诱导剂雷帕霉素作用于HEL细胞和PV患者骨髓细胞12、24、48 h后,各时间点细胞增殖活力与常规培养组相比明显增强,48 h时HEL细胞和PV患者骨髓细胞增殖活力分别为101 413±3 720和18 744±1 015;自噬抑制剂3-MA作用于HEL细胞和PV患者骨髓细胞12、24、48 h后,各时间点细胞增殖活力与常规培养组相比明显减弱,48 h时HEL细胞和PV患者骨髓细胞增殖活力分别为5 732±166和5 371±56.结论 JAK2V617F阳性HEL细胞和PV患者造血细胞存在较高的基础自噬水平,上调自噬活性可促进其增殖;下调自噬活性对其增殖有明显抑制作用.Objective To detect the activity of autophagy and explore the impact on survival and proliferation of HEL cells and hematopoietic cells of polycythemia vera （PV） patients with JAK2 V617F mutation.Methods Flow cytometry,AO staining and Western blot methods were used to detect the autophagy activity and the expression of LC3-Ⅱ protein of JAK2 V617F＋ HEL cells and hematopoietic cells of 12 newly diagnosed PV patients with JAK2 V617F mutation.HEL cells and bone marrow cells of 3 PV patients were treated with rapamycin or 3-MA to induce and inhibit autophagy,respectively.CellTiter Glo（R）method was used to detect the proliferation activity of cells.Results There was higher level of mean LC3-Ⅱ fluorescence intensity in HEL cells （159 389±29 001） than that in K562 cells （96 047±24 134） （P=0.044）.The formation of autophagosome in HEL cells is more than that in K562 cells detected by microscope.What＇s more,the level of mean LC3-Ⅱ fluorescence intensity in 12 PV patients＇ myeloid cells （92 842±4 250） was higher than that of 15 healthy volunteers （86 633±2 504） （P=0.001）.The expression of LC3-Ⅱ protein was higher in PV patients＇ peripheral blood cells than that in healthy volunteers detected by Western blot.After treated with rapamycin 12,24,48 h,the activity of autophagy in HEL cells and bone marrow cells of 3 PV patients were increased and the proliferation activity was higher than the control group,the proliferation activity at 48 h were （101 413 ±3 720）,（18 744± 1 015）,respectively.However,after treated with 3-MA 12,24,48 h,the activity of autophagy was decreased and the proliferation activity was lower than the control group,the proliferation activity at 48 h were （5 732±166）,（5 371 ±56）,respectively.Conclusions There is high basical activity of autophagy in JAK2 V617F＋ HEL cells and hematopoietic cells of PV patients with JAK2 V617F mutation.Up-regulated autophagy promotes proliferation of JAK2 V617F＋ HEL cells and bone marrow cells of PV patie

关 键 词：自噬 真性红细胞增多症 基因 JAK2V617F 细胞增殖 

分 类 号：R555.1[医药卫生—血液循环系统疾病]