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背景:中药3’-甲异靛玉红及,靛玉红衍生物在抗肿瘤的同时对机体正常免疫细胞影响的报道查及较少。目的:观察3’-甲异靛玉红对C57BL/6小鼠胸腺、脾淋巴细胞增殖的影响。设计、时间及地点:随机分组细胞病理学观察实验,于2007-08/2008-01在南方医科大学珠江医院完成。材料:实验动物为C57BL/6小鼠,雄性,6~8周龄,体质量(20±2)g。方法:取C57BL/6小鼠胸腺、脾组织研磨过滤为单细胞悬液,应用5,10,15,20,25μmol/L甲异靛玉红处理C57BL/6小鼠胸腺和脾细胞。以未经任何药物处理的C57BL/6小鼠作为空白对照,以给予刀豆蛋白A处理的C57BL/6小鼠为阳性对照。主要观察指标:①MTT法测定C57BL/6小鼠脾和胸腺淋巴细胞的增殖。②ELISA法测定培养上清中白细胞介素12活性。③流式细胞仪检测细胞周期、细胞凋亡率及死亡率,细胞内活性氧浓度。④RT-PCR法检测细胞bcl-2和cdk2mRNA水平。⑤荧光显微镜检测细胞Bcl-2、Bax和CDK2蛋白表达率。结果:15,20,25μmol/L甲异靛玉红作用24h均可明显抑制C57BL/6小鼠胸腺、脾淋巴细胞增殖(P<0.05),并呈量效、时效依赖性。各浓度的甲异靛玉红对C57BL/6小鼠胸腺和脾细胞各时间点分泌白细胞介素12的功能较对照组明显减低(P<0.05)。15μmol/L的3’-甲异靛玉红可导致C57BL/6小鼠胸腺、脾淋巴细胞凋亡相关蛋白bcl-2和细胞周期蛋白cdk2mRNA表达水平均减低,Bcl-2的蛋白表达水平降低,CDK蛋白表达减低,Bax表达水平升高,Bcl-2/Bax比例明显下降,启动细胞凋亡。15μmol/L的3’-甲异靛玉红将C57BL/6小鼠胸腺、脾淋巴细胞阻遏于G2/M期,细胞内活性氧水平呈量效、时效依赖性升高。结论:一定浓度范围甲异靛玉红可逆性抑制C57BL/6小鼠体外培养的脾及胸腺淋巴细胞增殖,同时抑制白细胞介素12的分泌,并启动细胞凋亡。
Background: The reports on the effect of 3’-meisoindigo and indirubin derivatives on the normal immune cells in the antitumor are rarely reported. Objective: To observe the effect of 3’-meisoindigo on the proliferation of thymus and spleen lymphocytes in C57BL / 6 mice. DESIGN, TIME AND SETTING: The randomized cytopathology observation experiment was performed at Zhujiang Hospital of Southern Medical University from August 2007 to January 2008. MATERIALS: The experimental animals were C57BL / 6 mice, male, 6-8 weeks old and body weight (20 ± 2) g. Methods: The thymus and spleen of thymus and spleen of C57BL / 6 mice were grinded and filtered into single cell suspension. The thymus and spleen cells of C57BL / 6 mice were treated with 5, 10, 15, 20, 25μmol / L meisoindigo. C57BL / 6 mice without any drug treatment served as blank control and C57BL / 6 mice treated with concanavalin A as positive control. MAIN OUTCOME MEASURES: ① The proliferation of spleen and thymus lymphocytes in C57BL / 6 mice was determined by MTT assay. ② ELISA assay of interleukin 12 activity in culture supernatant. ③ Flow cytometry to detect cell cycle, apoptosis rate and mortality, intracellular reactive oxygen species concentration. ④ RT-PCR assay of bcl-2 and cdk2 mRNA levels. ⑤ Fluorescence microscopy was used to detect the expression of Bcl-2, Bax and CDK2. Results: 24h, 24h after treatment with 15, 20 and 25μmol / L of methylisothiocyanine all inhibited the proliferation of thymus and spleen lymphocytes in C57BL / 6 mice significantly (P <0.05), and showed dose-dependent and time-dependent effects. The effect of each concentration of meisoindigo on the secretion of IL-12 in thymus and spleen cells of C57BL / 6 mice was significantly lower than that of the control group (P <0.05). 15μmol / L 3’-methylisoindil could cause the expression of bcl-2 and cyclin cdk2mRNA in thymus and spleen lymphocytes of C57BL / 6 mice to decrease and the protein expression of Bcl-2 to decrease, The expression of CDK decreased, the expression of Bax increased, the ratio of Bcl-2 / Bax decreased significantly, and the apoptosis was initiated. Thymus and spleen lymphocytes in C57BL / 6 mice were repressed in G2 / M phase by 15μmol / L 3’-methylisoindil. The level of reactive oxygen species increased in a dose-dependent manner and in a time-dependent manner. CONCLUSION: Methionine can reversibly inhibit the proliferation of spleen and thymus lymphocytes cultured in vitro in C57BL / 6 mice, inhibit the secretion of interleukin-12 and activate apoptosis in a certain concentration range.