c—Jun氨基末端激酶在小鼠T细胞增殖中的作用

Effect of c-Jun N-terminal kinase on the proliferation of murine T lymphocytes

目的研究c-Jun氨基末端激酶（c-Jun N-terminal kinase，INK）在小鼠T细胞增殖中的作用。方法以活体染料羧基荧光素乙酰乙酸琥珀酰亚胺酯（CFSE）染色，建立了在多克隆刺激剂刀豆蛋白A（ConA）刺激下评价小鼠T细胞增殖的模型，通过流式细胞术分析JNK的一种新的特异性抑制剂SP600125在不同剂量、不同时间对T细胞增殖的影响，并应用CellQuest软件分析增殖细胞各代所占比例和增殖指数（PI）及抑制指数（HI）。采用碘化丙锭染色分析不同剂量SP600125对ConA或佛波醇酯（PDB）加离子霉素（Ion）刺激的小鼠T细胞周期变化的作用。结果随着SP600125浓度从1．0μmol／L逐渐增至16．0μmol／L，ConA对T细胞的促增殖作用逐渐减弱，以8．0～16．0μmol／L的抑制作用最为明显，呈剂量依赖关系（r=0．98，P〈0．01）；选择最佳剂量8．0μmol／L，SP600125对T细胞增殖的抑制作用随时间从24h递增至96h而逐渐增强；进一步发现SP600125能使PDB加Ion刺激的小鼠T细胞停滞于G0/G1期，阻止其进入S期和c2，M期，且阻止作用随上述浓度的增加而增强，呈明显剂量依赖关系（S期：r=-0．98，P〈0．01；G2／M期：r=-0．88，P〈0．05）；SP600125也呈剂量依赖性地使ConA诱导的T细胞停滞于G0/G1期，阻止其进入S期（r=-0．90，P〈0．05）。结论JNK信号通路的活化在小鼠T细胞增殖中可能起着重要作用。

Objective To investigate the effect of c-Jun N-terminal kinase（JNK） on the proliferation of T lymphocytes of mice. Methods A model to evaluate T cell proliferation stimulated with a polyclonal activator, concanavalin A（ Con A）, was established by vital dye carhoxyl fluorescin diacetate suecinmidyl ester（CFSE） labeling technique. Effects of a new and specific inhibitor for JNK （SP600125）, on T cell proliferation at different doses and in different time were estimated by flow cytometry. At the same time, the percentage of proliferating T cells, proliferation index（M） and inhibition index（HI） were analyzed by CollQuest software. The propidium iodide labeling technique was applied to assay the effect of different doses of SP600125 on change of T lymphocyte cell-cycle stimulated by Con A or by phorhol ester（PDB） plus ionomycin （Ion）. Results CFSE staining showed that T cell proliferation was decreasing with the concentration of SP600125 arising from 1.0 μmol/L to 16.0 μmol/L. At the range of 8.0 μmol/L to 16.0 μmol/L, the inhibitory effect was the most significant, suggesting a dose-dependent manner（r = 0.98,P 〈0.01）. At the dose of 8.0 μmol/L, the inhibitory effect on the T cell proliferation was enhanced with incubation elongated from 24 h to 96 h. The analysis of the cell-cycle distribution revealed that SP600125 led to G0/G1 arrest and blocked S and G2/M phase entry stimulated by PDB plus Ion in a dose-dependent manner（S phase： r = -0.98, P 〈0.01; G2/M phase： r = -0.88, P 〈0.05）. SP600125 could also lead to G0/G2 arrest and block T cells to enter S phase stimulated by Con A in a dose-dependent manner（ r = - 0.90, P 〈 0.05）. Conclusion The activation of JNK signaling pathway may play an important role in T lymphocyte proliferation.