细胞外信号调节激酶1/2的阻断剂PD98059对小鼠淋巴细胞增殖的影响

目的探讨细胞外信号调节激酶1/2(ERK1/2)信号通路的特异性阻断剂PD98059对小鼠淋巴细胞体外增殖的影响。方法分离小鼠淋巴结细胞,藉羧基荧光素乙酰乙酸(CFDA-SE)染色,用多克隆刺激剂刀豆A蛋白(ConA)或者佛波醇酯(PDB)加离子霉素(Ion)刺激,以流式细胞术分析PD98059对淋巴细胞增殖的影响;利用碘化丙锭(PI)染色分析PD98059对细胞周期的影响。结果CFDA-SE染色分析显示,当浓度为5、10、20、30、40μmol/L时,PD98059对ConA诱导的淋巴细胞增殖具有明显的抑制作用,并呈现明显的剂量依赖关系(r=0.985,P<0.01)。选择最佳浓度30μmol/L的PD98059,观察其对不同时间淋巴细胞增殖的影响,结果发现,随着时间的延长,PD98059对淋巴细胞增殖的抑制作用明显降低(P<0.01),但抑制率随时间的延长而明显升高。细胞周期分析进一步表明,PD98059可阻止ConA刺激的淋巴细胞进入S期和G2/M期,而亚二倍体峰变化并不明显。PD98059对PDB Ion刺激的淋巴细胞细胞周期的影响与ConA刺激相似,不同的是S期和G2/M期的变化较ConA的作用更明显。结论PD98059对小鼠淋巴细胞的增殖有明显地抑制作用,并可阻止其进入S期和G2/M期,提示ERK1/2信号通路的活化在淋巴细胞增殖中起重要作用。

Effect of extracellular-signal regulated kinase 1/2 specific repressor PD98059 on murine lymphocyte proliferation

AIM: To investigate the effect of PD98059, a specific repressor of extracellular signal regulated kinase 1/2 (ERK1/2), on the proliferation of the murine lymphocytes. METHODS: Lymphocytes were isolated from lymphoid nodes of BALB/c mouse. After stained with carboxy fluorescein diacetate, succinimidyl ester(CFDA-SE), the cells were stimulated with polyclonal activators, concanavalin(ConA) and phorbol 12,13-dibutyrate(PDB) plus ionomycin(Ion). The proliferation and cell-cycle of lymphocytes were analyzed using flow cytometry(FCM). RESULTS: CFDA-SE staining showed that 5, 10, 20, 30 and 40 micromol/L PD98059 could significantly inhibit the proliferation of murine lymphocytes induced by ConA. The inhibitory effect was dose-dependent(r=0.985, P<0.01). The optimal concentration of 30 micromol/L PD98059 was selected and its inhibitory effect was significantly decreased with prolongation of time (P<0.01); but the inhibitory rate increased. Analysis of the cell cycle induced by ConA revealed that the cells was blocked into S and G2/M phases as the concentration of PD98059 increased. However, the cell number in sub-G0/G1 peak (apoptotic peak) had no significant change under the concentration range used. PD98059 had the similar effect on the cells treated with PDB plus Ion, but S and G2/M phases were more distinct. CONCLUSION: PD98059 can inhibit the proliferation of the murine lymphocytes and blocked them into S and G2/M phases, indicating the activation of ERK1/2 signaling pathway plays an important role in the proliferation of lymphocytes.