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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
Objective To explore the effect of mitogen activated protein kinase (MAPK) signaling inhibitor on the proliferation and differentiation of UT7 cells induced by megakaryocyte growth factor (TPO), and to elucidate the mechanism of TPO on UT7 cells. Methods EGFPpMSCV and MEK1pMSCV plasmids were transfected into UT7 cells. The transfected cells were co-cultured with TPO. The phosphorylation of MEK1 (MAPK / Erkkinase1) in UT7 cells was detected by Western blot. The effect of MEK1, a MAP2 / The effect of MEK1 with transfection mutation (Ser222A) and PD98059, a MAPK blocker, on CD41 expression in UT7 cells was detected by flow cytometry. Results ① The recombinant retroviral vectors MEK1pMSCV and EGFPpMSCV were transfected into UT7 cells. The positive rate of EGFP was 60.73%. (2) TPO different role of time (1h, 3h), the experimental group phosphorylated MEK1 were lower than the control group (P value <0.05). (3) MAPK inhibitor PD98059 and exogenous MEK gene blocked the proliferation of UT7 cells induced by TPO. The cell proliferation rate of DMSO control group was 98.58%, and the cell proliferation rate of PD98059 group was 39.00% (P <0.05) The cell proliferation rate was 102.12% in EGFPpMSCV group and 48.93% in MEK1pMSCV group (P <0.05). (4) The expression of CD41 on UT7 cells (10.27%) in MAPK inhibitor PD98059 group was significantly lower than that in control group (36.43%). CD41 expression in UT7 cells transfected with MEK1pMSCV was significantly lower than that in transfected cells (24.03%, 40.16%). Conclusion TPO induces the phosphorylation of MEK1 in UT7 cells. The time course of TPO is obviously related to the phosphorylation of MEK1 in UT7 cells. The MAPK signal transduction pathway mediates TPO to promote UT7 cells