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目的探讨人肝再生增强因子(hALR)对人外周血单个核细胞(PBMC)增殖的影响及其机制。方法梯度离心分离PBMC,将细胞分为正常对照组、刀豆蛋白A(ConA)(5mg/L)组和ConA(5mg/L)+hALR(30mg/L)组,分别培养10min、30min、1h、2h、4h后,MTT法检测各组各时间点细胞增殖水平,钙荧光指示剂法检测细胞内游离Ca2+浓度的变化,Western blot法检测细胞内胞外信号调节激酶(ERK)的磷酸化程度。结果4h内,各组细胞的增殖水平差异无统计学意义,但ConA组细胞增殖水平已表现出逐渐升高的趋势。正常对照组细胞内Ca2+浓度在加入Fluo-3/AM后2h达高峰,ConA组1h达高峰,ConA+hALR组10min达高峰。正常对照组细胞内磷酸化的ERK随培养时间的延长而逐渐减少;ConA组细胞内磷酸化的ERK在30min达高峰,之后逐渐降低;ConA+hALR组细胞内磷酸化的ERK在2h前明显低于ConA组。结论hALR可能通过影响细胞内Ca2+浓度的变化峰值和磷酸化的ERK,来抑制人PBMC的免疫功能,从而影响其增殖。
Objective To investigate the effect of human augmenter of liver regeneration (hALR) on the proliferation of human peripheral blood mononuclear cells (PBMC) and its mechanism. Methods PBMCs were separated by gradient centrifugation and divided into normal control group, ConA (5mg / L) group and ConA (5mg / L) + hALR group (30mg / L) for 10min, 30min, , 2h, 4h later, the cell proliferation was measured by MTT assay, the intracellular free Ca2 + concentration was detected by calcium fluorescence indicator, and the phosphorylation of extracellular signal-regulated kinase (ERK) . Results Within 4h, there was no significant difference in the proliferation of cells in each group, but the cell proliferation in ConA group showed a gradual increase trend. The intracellular Ca2 + concentration in normal control group peaked at 2h after addition of Fluo-3 / AM, peaked at 1h in ConA group and peaked at 10min in ConA + hALR group. The phosphorylated ERK in control group decreased gradually with the prolongation of culture time. The phosphorylation of ERK in ConA group peaked at 30 min and then decreased gradually. The phosphorylation of ERK in ConA + hALR group was significantly lower 2h In ConA group. Conclusion hALR may affect the immune function of human PBMC by influencing the peak of intracellular Ca2 + concentration and phosphorylation of ERK, thereby affecting its proliferation.