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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。
This study was to investigate the effects of evodiamine on proliferation, apoptosis and biological function of thymocytes and spleen cells in different germline mice. 8-week-old male BALB/c, C57BL/6 and F1 hybrid mice were sacrificed, and their thymus and spleen were used to prepare a single cell suspension. The proliferation of spleen and thymocytes induced by ConA was measured by MTT assay. The activity of IL-2 in culture supernatant was determined by ELISA. The mRNA levels of bcl-2 and cdk2 in 0.75 μmol/L evodiamine-treated cells were measured by RT-PCR. Cytometry was used to measure cell cycle and apoptosis rate. Intracellular reactive oxygen species (ROS) levels were detected indirectly by percentage of cells positive for dihydrodichlorofluorescein (DCF). BCL-2 and CDK2 were detected by fluorescence microscopy. , BAX protein expression rate. The results showed that when the concentration of evodiamine ≥ 0.5 μmol/L, the proliferation of thymocytes and spleen cells induced by ConA was inhibited at 24, 48 and 72 hours (p<0.05). The function of releasing IL-2 from mouse spleen cells and thymocytes was significantly lower than that of the control group (p<0.05), and the mRNA levels of bcl-2 and cdk2 were significantly decreased after treatment with evodiamine 0.75 μmol/L. Compared with the control group, the thymocytes and spleen cells of three kinds of mice could be induced apoptosis with evodiamine 0.75μmol/L treatment for 6, 12, 24 hours (p<0.05). The number of DCF-positive cells in the 12-hour drug group was significantly higher than that in the control group. The percentage of DCF-positive cells in the drug group was not significantly different from that in the control group at 24 and 48 hours. However, two groups of cells with different sizes appeared in the drug-treated group, and the histogram showed double peaks. Among them, a group of cells showed negative DCF, indicating that evodiamine increased the intracellular ROS level, but as the drug treatment time prolonged, the ROS level in the dying cells was significantly reduced. After evodiamine treatment, the expression of BCL-2 and CDK2 protein was significantly decreased, and the expression of BAX protein was up-regulated. Conclusion: Evodiamine can inhibit the proliferation of splenocytes and thymocytes in vitro by inducing bcl-2 and cdk2 down-regulation and induce their apoptosis. The activity of IL-2 secreted by cells is also decreased.