【摘要】 目的探索促分裂原活化蛋白激酶(mitogenactivatedproteinkinase,MAPK)信号阻断剂对巨核细胞生长分化因子(TPO)促进UT7细胞增殖和分化作用,阐明TPO对UT7细胞作用的机制。方法将EGFPpMSCV和MEK1pMSCV质粒转染UT7细胞;转染细胞与TPO共培养,Westernblot法检测UT7细胞MEK1(MAPK/Erkkinase1)磷酸化;观察转染突变(Ser222A)的MEK1和MAPK阻断剂PD98059对UT7细胞增殖的影响;采用流式细胞仪检测转染突变(Ser222A)的MEK1和MAPK阻断剂PD98059对UT7细胞表达CD41的影响。结果①重组逆转录病毒载体MEK1pMSCV和EGFPpMSCV转染UT7细胞,EGFP阳性率为60.73%。②TPO不同的作用时间(1h,3h),实验组磷酸化MEK1均低于对照组(P值均<0.05)。③MAPK阻断剂PD98059和外源突变MEK基因阻断了TPO对UT7细胞的增殖作用,DMSO对照组的细胞增殖率为98.58%,PD98059组的细胞增殖率为39.00%(P<0.05);转染EGFPpMSCV组细胞增殖率为102.12%,转染MEK1pMSCV组细胞增殖率为48.93%(P<0.05)。④MAPK阻断剂PD98059组UT7细胞CD41表达(10.27%)明显低于对照组(36.43%),转染MEK1pMSCV组UT7细胞CD41表达(24.03%)明显低于转染EGFPpMSCV组(40.16%)。结论TPO诱导UT7细胞MEK1磷酸化,TPO作用时间与UT7细胞MEK1的磷酸化有明显的关系,MAPK信号转导通路介导TPO促进UT7细胞更多还原

【Abstract】 Objective To explore the effects of MAPK antagonist on TPO stimulated UT7 cell proliferation and differentiation, and to elucidate the mechanism of TPO functioning on UT7 cells. Methods EGFP pMSCV and MEK 1 pMSCV MEK 1 plasmids were transferred into UT7 cells. Phosphorelated MEK1 of UT7 cells was examined by Western blot. The proliferation and CD41 expression of UT7 cells transfected with mutant (ser222A) MEK1 or exposed to PD98059 were examined. Results ① 60.73%EGFP positive cells were obtained in retroviral vector MEK1 pMSCV transfected UT7cells. ② In different time of TPO stimulating UT7 cells, the level of phosphorelated MEK1 was lower in experiment group than in control group. In experiment group, the level of phosphorelated MEK1 was decreased after stimulated by TPO for 1 hour,and almost disappeared after stimulated for 3 hours ③ The effect of TPO on UT7 cell proliferation was inhibited by PD98059 and the transfected mutation MEK1 gene. The proliferation rate was 98.58% in DMSO control group, 39.00% in PD98059 group (P<0.05), 102.13% in EGFP pMSCV group, and 48.94% in MEK1pMSCV(P<0.05).④ The CD41 expression on UT7 was inhibited by PD98059 and the transfected mutation MEK1 gene. Conclusion Phosphorelation of MEK1 in UT7 cells can be induced by TPO. There was a relationship between the TPO stimulating time and phosphorelation of MEK1. The effects of TPO on UT7 cell proliferation and CD41 expression is mediated by MAPK signal transduction pathway.更多还原