吴茱萸碱对不同种系小鼠免疫功能的调节(英文)

Immunoregulatory Effect of Evodiamine in Mice of Various Germlines

本研究探讨吴茱萸碱对不同种系小鼠胸腺细胞和脾细胞的增殖、凋亡及生物功能的影响。处死8周龄雄性BALB/c、C57BL/6及F1代杂交鼠,取其胸腺、脾制备单细胞悬液。用MTT法测定ConA诱导后脾和胸腺细胞增殖活性,用ELISA法测定培养上清IL-2活性,RT-PCR检测0.75μmol/L吴茱萸碱处理的细胞bcl-2和cdk2基因mRNA水平,流式细胞仪检测细胞周期和细胞凋亡率,以双氢二绿荧光黄(2 7-dichlorofluorescein,DCF)阳性的细胞百分比间接检测细胞内活性氧(ROS)水平,应用荧光显微镜检测BCL-2、CDK2、BAX蛋白表达率。结果表明:当吴茱萸碱浓度≥0.5μmol/L时,在24、48和72小时3种小鼠ConA诱导的胸腺细胞、脾细胞的增殖均被抑制(p<0.05),吴茱萸碱处理的各种系小鼠脾细胞和胸腺细胞释放IL-2的功能较对照组明显减低(p<0.05),吴茱萸碱0.75μmol/L处理后细胞bcl-2和cdk2的mRNA水平较对照组明显降低。与对照组相比,吴茱萸碱0.75μmol/L处理6、12、24小时均可诱导3种小鼠胸腺细胞、脾细胞明显凋亡(p<0.05)。12小时药物组DCF阳性细胞均明显高于对照,24和48小时药物组DCF阳性的细胞百分比与对照组无明显差异,但药物处理组细胞出现大小不一的两群细胞,直方图呈双峰,其中一群细胞DCF呈阴性,这表明吴茱萸碱使细胞内ROS水平增高,但随着药物处理时间的延长,濒死细胞内ROS水平明显降低。吴茱萸碱处理后BCL-2、CDK2蛋白表达明显降低,BAX蛋白表达上调。结论:吴茱萸碱可通过bcl-2、cdk2下调,抑制多种系小鼠体外培养的脾细胞和胸腺细胞增殖并诱导其凋亡,同时细胞分泌IL-2的活性亦明显下降。

The aim of this study was to investigate the effect of evodiamine on the proliferation and the immune function of thymocytes and splenocyte of mice from three germlines, which were 8 weeks old masculinity BALB/c, C57BL/ 6 and F1 hybridization mice. Cells of thymus and spleen were harvested and prepared as unicellular suspension. Cell proliferation was detected by MTT method, while the concentration of IL-2 was detected by ELISA, mRNA levels of bcl-2 and cdk2 in cells treated with evodiamine were detected by RT-PCR, the apoptosis rate and intracellular reactive oxygen species （ROS） concentration were amalyzed by FCM, and the protein levels of BCL-2, CDK2 and BAX were determined by fluorescence microscope. The results indicated that at 0.5, 0.75 and 1 μmol/L, evodiamine inhibited the proliferation and externalization of thymocytes and splenocytes stimulated with ConA （p 〈0.05）. At 0.75 μmol/L, evodiamine inhibited the secretion of IL-2 ,decreased the mRNA level of bcl-2 and cdk2, and induced apoptosis of thymocytes and splenocytes（p 〈0.05 ）. Intracellular ROS concentration increased significantly after treatment with evodiamine for 12 hours （p 〈0.05）. The death rate increased at a prolong period of time. After treatment with evodiamine for 24 and 48 hours, the cells were divided into two groups, one of which was negatively stained by 2 7-dichiorofluorescein（ DCF）, which indicated that ROS level decreased significantly in the dying cells. It is concluded that evodiamine inhibits proliferation and induces apoptosis of thymocytes and splenocytes from different germline mice, and at the same time decreases secretion of IL-2 through down-regulating bcl-2 and cdk2 levels.