ERK1/2信号通路阻断剂PD98059对慢性高原病患者骨髓有核红细胞中Ras、BRaf、MEK、ERK1/2表达的影响

Effect of ERK1/2 Signaling Pathway Inhibitor PD98059 on the Expression of Ras,BRaf,MEK,ERK1/2 in Marrow Nucleated Red Blood Cells of CMS Patients

目的:探讨ERK1/2信号通路阻断剂PD98059对Ras、B型丝/苏氨酸蛋白激酶(BRaf)、丝裂原活化蛋白激酶(MEK)、细胞信号调节激酶1/2(ERK1/2)表达的影响,以期为慢性高原病(chronic mountain sickness,CMS)的基础研究和临床治疗探讨新的途径。方法:选取CMS患者16例,取骨髓液分离单个核细胞,以CD71与CD235a抗体磁珠分选阳性细胞,将细胞分为5组:空白对照组、DMSO溶剂组及PD98059 5、10和20μmol/L组。在低氧条件下培养72 h,应用ELISA法测定培养骨髓有核红细胞上清液中Ras-GTP水平,RT-PCR法测定骨髓有核红细胞中BRaf、MEK、ERK1/2 mRNA的表达,Western blot方法检测骨髓有核红细胞中p-BRaf、p-MEK、pERK1/2蛋白表达。结果:PD98059对各组Ras-GTP的水平无明显影响(P=0.798)。溶剂组与空白对照组相比,BRaf、MEK mRNA的表达水平差异无统计学意义(P=0.826、P=0.298)。与PD98059 20 mol/L比较,其余4组ERK1/2 mRNA的表达水平差异有统计学意义(P=0.001、P=0.002)。溶剂组与空白对照组相比,p-BRaf、p-MEK蛋白的表达差异无统计学意义(P=0.370、P=0.351)。与PD98059 20 mol/L比较,其余4组p-ERK1/2蛋白水平差异有统计学意义(P<0.001、P<0.007)。结论:PD98059能下调骨髓有核红细胞ERK1/2 mRNA及p-ERK1/2蛋白的表达。Ras/Raf/MEK/ERK 1/2通路是调控CMS骨髓有核红细胞的主要信号传导途径,可能参与了慢性高原病的发病过程。

Objective： To investigate the effect of ERK1/2 signaling pathway inhibitor PD98059 on Ras,Raf,MEK,ERK1,ERK2 expression in order to explore a new way for basic research and clinical treatment of the chronic mountain sickness（CMS）. Methods： Sixteen CMS patients were selected,the bone marrow was collected for isolation of monomuclear cells（MNC）,the cells were sorted by using CD71 and CD235 a antibody magnetic beads,then positive cells were diveded into 5 groups： blank control,DMSO and PD98059 5,10 and 20 μmol/L,and were cultured in hypoxid condition for 72 hours. The Ras-GTP levels in supernatant was detected by ELISA,the RT-PCR was used to determine the expression of BRaf,MEK,ERK1,ERK2 mRNA in nucleated red blood cells,and the Western blot method was used to detect expression of BRaf,MEK,ERK1,ERK2 protein. Results： PD98059 had no effect on the level of Ras-GTP in each groups. Compared with the blank control group,the expression levels of BRaf,MEK mRNA in DMSO group were not statistically significant（P values were 0. 826,0. 298）. Compared with the PD98059 20 mol/L group,the expression level of ERK1/2 mRNA was statistically significant（P = 0. 001,0. 002）. Compared with the blank control group,expression levels of p-BRaf,p-MEK protein in DMSO group were not statistically significant（P =0. 370,0. 351）. Compared with the PD98059 20 mol/L group,the difference of p-ERK1/2 protein level in other 4 groups were statistically significant（P values were 0. 001,0. 007）. Conclusion：PD98059 can up-regulate the expressions of ERK1/2 miRNA and p-ERK1/2 protein in bone marrow nucleated red blood cells,the Ras/Raf/MEK/ERK 1/2 pathway is the main signal transduction pathway in regulating bone marrow nucleated red blood cells,suggesting that Ras/Raf/MEK/ERK 1/2 pathway may be involved in the pathogenesis of chronic mountain sickness process.