环孢菌素A对小鼠T细胞活化影响的血清免疫药理学评价

Serum immunopharmacological assessment of effects of cyclosporine A on the activation of murine T cells

目的 应用血清免疫药理学技术 ,探讨环孢菌素A(CsA)对T细胞体外活化的影响 ,以进一步揭示CsA免疫调节的分子机制 ,为临床用药提供理论依据。方法 分离小鼠淋巴结细胞 ,分别与CsA、5 %CsA血清预孵后 ,加入多克隆刺激剂继续培养共 2 4h。收获细胞 ,进行双色免疫荧光标记 ,以流式细胞术分析T细胞上CD6 9分子的表达情况。结果 在CsA和 5 %CsA血清作用下 ,ConA活化的T细胞CD6 9表达的百分率分别为 (2 4 15± 3 38) %和 (2 3 6 8±6 89) % ,与相应对照组 [分别为 (6 4 6 7± 5 88) %和 (6 4 35± 10 13) % ]相比较均有显著差异 (P <0 0 1) ;在CsA和5 %CsA血清作用下 ,PDB活化的T细胞CD6 9表达的百分率分别为 (78 86± 7 70 ) %和 (80 0 5± 9 91) % ,与相应对照组 [分别为 (84 79± 6 87) %和 (79 6 8± 4 17) % ]相比较均无显著差异 (P >0 0 5 )。结论 CsA(410nmol/L)和含CsA血清均可强烈抑制ConA刺激的T细胞活化 ,而对PDB刺激的T细胞活化无抑制效应。 5 %CsA血清与CsA单纯药物体外实验的结果相一致 。

Aim To explore the effects of cyclosporine A on the T cells activated by mitogen so as to provide immunopharmacological basis for the application of this drug clinically. Methods After separation of the lymphocytes from lymphoid nodes of C57BL/6 mouse, these cells were preincubated with CsA and CsA containing serum respectively for a hour,and then further incubated with polyclonal activators(Con A or PDB) for 24 hours. Harvesting the cells, the expression rate of CD69 on T cells was analyzed by flow cytometry following two color immunofluorescent staining. ResultsThe expression rates of CD69 on the T cells activated by Con A under the action of CsA and CsA containing serum were 24 15% and 23 68% respectively, having significant difference as compared with the expression rate of the control groups ( P <0 01,being 64 67%±5 88% and 64 35%±10 13%, respectively),Similarly, the expression rates of CD69 on the T cells activated by PDB under under like conditions were (78 86±7 70)% and (80 05±9 91)% respectively, which all was not significantly differently from the results of the control groups ( P >0 05). Conclusion Both CsA(410 nmol/L)and CsA containing serum showed inhibitory effects on the activation of T cells in response to stimulation of Con A except for PDB. Our data show that the immunosuppressive effect of CsA is through the calcium dependant signaling pathway during activation of T cells, and it has no relevance to PKC θ signaling pathway. The result of CsA containing serum is in great concordance with that of CsA in vitro in our model,which reflect the effectiveness and feasibility of the assessment of serum immunopharmacology to a certain extend.