莫诺苷对小鼠T细胞体外活化与分化的影响

Effect of Morroniside on the activation and differentiation of T lymphocytes in mice

目的:探讨山茱萸提取物莫诺苷对小鼠T细胞功能的影响及可能的机制。方法:将C57BL/6小鼠脾脏制成单细胞悬液,采用抗CD3、抗CD28功能抗体模拟抗原刺激活化T细胞,分为对照组(不做任何处理)和莫诺苷组(10μmol/L莫诺苷处理)。采用流式细胞术分析CD4^+T和CD8^+T细胞表面分子CD69、CD25表达水平,以及其增殖、凋亡与分化为Th1、Th2、Treg等细胞的情况;利用实时荧光定量PCR检测细胞中Tbx21、Gata3、淋巴样增强因子1(lymphoid enhance factor 1,LEF1)等mRNA水平;应用ELISA法测定培养上清液中IFN-γ、IL-4以及TGF-β含量;运用蛋白质免疫印迹技术测定细胞β-连环蛋白表达量。结果:与对照组相比,莫诺苷组中活化T细胞向Th1、Th2、Treg分化比例增加(P <0. 05),CD25活化标志增强,T细胞增殖标志Ki67有所增强,但增殖和凋亡差异均无统计学意义;IL-4和Treg细胞相关的TGF-β因子水平增加(P <0. 05),但Th1细胞分泌的IFN-γ差异无统计学意义;Tbx21、Gata3、LEF1等mRNA水平均增加(P <0. 05);蛋白质印迹显示β-连环蛋白表达明显上调(P <0. 01)。结论:山茱萸提取物莫诺苷可促进小鼠T细胞分化,其机制可能与Wnt信号通路有关。

Objective: To study the effect of Cornus officinalis extract Morroniside on T lymphocytes and its potential mechanisms. Methods: The spleen of C57 BL/6 mice was used to prepare single cell suspension,which were activated with anti-CD3 and anti-CD28 stimulation. Splenocytes were divided into control group and Morroniside group( treated with 10 μmol/L Morroniside). The activation( CD69,CD25),proliferation and apoptosis in both CD4+and CD8+T cells were detected. Moreover,the differentiation proportions of CD4+T cell subsets were analyzed by flow cytometry. CD4+T cell subsets were analyzed by flow cytometry. The mRNA level of Tbx21,Gata3,lymphoid enhance factor 1( LEF1) in cells were detected by real-time fluorescence quantitative PCR. The protein expression of β-catenin was analyzed by Western blotting. Results: Compared with the control group,the proportions of activated T lymphocytes differentiated into Th1,Th2 and Treg were significantly increased in the morroniside group( P < 0. 05). Although the activation and proliferation marker CD25 and Ki67 were enhanced,there were no significant difference in the proportions of proliferation and apoptosis between the two groups. The cytokine levels of IL-4 secreted by Th2 cells and TGF-β related to Treg cells were also markedly increased( P < 0. 05). IFN-γ secreted by Th1 cells was not statistically significant. The mRNA levels of Tbx21,Gata3,LEF1 and the protein expression of β-catenin were increased in the Morroniside group( P <0. 05). Conclusion: Morroniside extracted from Cornus officinalis could promote the differentiation of T lymphocytes,which may be related with Wnt signaling pathway.