目的:研究C2-神经酰胺(C2-cer)对小鼠T细胞体外活化和增殖的影响,并对其免疫调节作用进行初步探讨。方法:分离小鼠淋巴结细胞,加入刀豆蛋白A(ConA)或佛波醇酯(PMA)和离子霉素(Ion)进行刺激,以不同终浓度的C2-cer与T细胞共培养。流式细胞术结合双色荧光抗体染色,检测T细胞早期活化抗原CD69的表达;用羧基荧光素双醋酸盐琥珀酰(CFDA-SE)染色结合流式细胞术,并用ModF it软件分析T细胞增殖相关指数。结果:终浓度为25、50和75μmol/L的C2-cer可以显著抑制ConA诱导的T细胞CD69的表达,由ConA组的(60.13±1.18)%,分别降低为(54.56±1.14)%、(48.73±1.26)%和(27.09±1.07)%(P〈0.01);CFDA-SE染色结果显示,各浓度C2-cer对ConA诱导的小鼠淋巴细胞增殖,具有明显的抑制作用,增殖指数(PI)由ConA组的(1.81±0.25),分别降低为(1.46±0.01)、(1.25±0.04)和(1.18±0.03)(P〈0.05);各浓度C2-cer均可明显抑制PMA+Ion诱导的小鼠淋巴细胞增殖,增殖指数(PI)由PMA+Ion组的(1.47±0.01),分别降低为(1.27±0.01)、(1.11±0.01)和(1.05±0.01)(P〈0.05)。结论:C2-cer能有效地抑制小鼠T细胞的体外活化和增殖。
Aim:To investigate the effects of C2-ceramide on activation and proliferation of murine T lymphocytes, and to elucidate the mechanism of the immunosuppressive effect of C2-cer. Methods: Lymphocytes were isolated from lymphoid nodes of mouse, and were stimulated with polyelonal activators ConA or PMA plus Ion, and then were co-cultured with C:cer of different final concentration. The expression of CD69 (the early marker of the activated T ceils) on T lymphocytes were measured by flow cytometry combined with two colored monoclonal antibodies. While, the effect of C2-cer on the proliferation of T lymphocytes in response to ConA or PMA plus Ion stimulation was determined by flow cytometry combined with carboxyl fluorescein-diacetate-succinimidyl ester (CFDA-SE) staining technology, and the proliferation index (PI) was analyzed by ModFit software. Results: C2-cer ( final concentration is 25, 50 and 75 μmol/L) can inhibit the expression of CD69 on activated T lymphocytes in a dose-dependent manner (P 〈0.01). The expression rate of CD69 on T cells in response to ConA was (60. 13 ± 1.18)%. After treatment with C2-cer, the expression rate of CD69 reduced to (54. 56 ± 1.14) %, (48.73 ± 1.26) % and (27.09 ± 1.07) % respectively ( P 〈 0. 01 ) ; C2-cer can inhibit the proliferation of T lymphocytes stimulated by ConA at all experimental concentration. The proliferation index (PI) of T ceils in ConA group is ( 1.81 ±0.25), and the PI value of T cells in C2-cer group is ( 1.46 ±0. 01 ), ( 1.25 ±0. 04) and ( 1.18 ±0. 03), respectively (P 〈0. 05) ; C2-cer can also inhibit the proliferation of T lymphocytes stimulated by PMA ± Ion at all experimental concentration. The proliferation index (PI) of T cells in PMA + Ion group is ( 1.47 ± 0. 01 ), and the PI value of T cells in C2-cer group is ( 1.27 ± 0. 01 ), ( 1.11 ± 0. 01 ) and ( 1.05 ± 0. 01 ), respectively ( P 〈 0. 05 ). Conclusion: C2-cer can effectively in