C_2-神经酰胺对小鼠T淋巴细胞体外活化和增殖的影响

Effects of C_2-ceramide on activation and proliferation of mouse T lymphocytes in vitro

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 inhibit the activation and proliferation of mouse T lymphocytes in vitro.