目的:探讨八肽胆囊收缩素(CCK8)与表皮生长因子(EGF)在神经元信号转导中的交叉对话(cross-talk)及可能存在机制,并对CCK8与EGF的协同作用进行初步研究.方法:CCK8刺激表皮生长因子受体(EGFR)磷酸化的受体类型分析:培养的乳小鼠神经元随机分为对照组(等量的DMEM培养基)、CCK8刺激组(10-7 mol/L)、CCKA受体拮抗剂L364 718组、CCKB受体拮抗剂L365 260组以及L364 718+L365 260联合组(浓度均为10-8 mol/L),拮抗剂加入10 min后再给予CCK8,作用5 min后收集细胞提取蛋白,Western blotting检测磷酸化表皮生长因子受体(p-EGFR)蛋白表达量的变化;CCK8与EGF对神经元EGFR磷酸化状态的影响和CCK8与EGF协同作用功效分析:培养的乳小鼠神经元随机分为对照组、CCK8(10-7 mol/L)组、EGF(40 μg/L)组以及CCK8+EGF组,药物作用5 min后终止反应,处理和分析方法同前,同时通过MTT法检测各组神经元培养不同时间(24、48、72、96 h)后细胞的生长活性.结果:(1)2种CCK受体拮抗剂均可降低EGFR蛋白的磷酸化水平,其中A型受体拮抗剂的作用明显强于B型,2种拮抗剂同时作用对EGFR蛋白磷酸化水平的降低作用更为明显;(2)CCK8和EGF分别刺激神经元后,EGFR的磷酸化水平均增强,但CCK8+EGF刺激后表现更为显著;(3)CCK8与EGF联用对增强神经元生长活性和延长其存活时间的作用较两药单独作用效果更为明显(P<0.05).结论:(1)CCKA和CCKB受体均介导了CCK8激活EGFR磷酸化的信号转导途径,但A型受体发挥了更为重要的作用;(2)CCK8与EGF信号转导途径之间存在着cross-talk;(3)CCK8与EGF可能通过信号转导的cross-talk协同促进小鼠神经元的生长与存活.%AIM: To explore the mechanism of signaling transduction and cross talk between cholecystokinin octapeptide ( CCK8 ) and epidermal growth factor ( EGF ) in mouse neurons and to observe the effect of CCK8 in coordination with EGF on neuron growth and cell viability.METHODS: For determining which kind of CCK receptor mediated the phosphorylation of EGF receptor, the cultured neurons were randomly divided into control group, CCK8 stimulation group, CCKA receptor antagonist group, CCKB receptor antagonist group, and CCKA + CCKB receptor antagonist group.Control and stimulation groups were stimulated with DMEM and CCK8( 10-7 mol/L ) for 5 min, respectively, while antagonist groups were pre - incubated with different types of receptor antagonists (10 mol/L ) for 10 min and followed by stimulating the neurons with CCK8.For observing the effect of CCK8 and EGF on the phosphorylation of EGFR in neurons and on neuron growth and cell viability, the cultured neurons were randomly divided into control group, CCK8 stimulation group, EGF stimulation group and CCK8 + EGF stimulation group, which were stimulated with DMEM, CCK8( 10-7 mol/L), EGF ( 40 μg/L ) and CCK8 + EGF for 5 min, respectively.Reactions were terminated by freezing the neurons in liquid nitrogen and the phosphorylated EGFR was detected by Western blotting.Meanwhile, the viability of the neurons was observed by MTT method after stimulated for 24 h, 48 h, 72 h and 96 h.RESULTS: The phosphorylation levels of EGFR were decreased in the neurons treated with either of the two CCK receptor antagonists, and more obvious decrease was observed when the two CCK receptor antagonists were used in combination.Compared with control group, the phosphorylation levels of EGFR in the neurons were significantly increased( P ＜ 0.05 ) after stimulated with CCK8 or EGF, and the increase was more remarkable in CCK8 +EGF stimulation group.CCK8 or EGF improved the viability and prolonged the life span of the neuron, and synergism of these two reagents was observed.CONCLUSION: Both CCKA and CCKB receptors are involved in the phosphorylation of EGFR in the neurons stimulated by CCK8 , and the type A receptor may play a more important role.There is cross - talk between CCK8 and EGF signaling pathways in neurons.The signaling cross - talk between CCK8 and EGF may be the underlying molecular mechanism responsible for the synergistic effect on the neuron growth and viability in vitro.
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