氧化苦参碱对小鼠树突状细胞成熟和功能的影响

【作者】 张维；

【导师】 周伯平；

【作者基本信息】 湖北中医学院 ， 中西医结合临床， 2007， 硕士

【摘要】 【目的】研究氧化苦参碱对小鼠树突状细胞成熟、表型及功能的影响。【方法与分组】采用细胞因子小鼠集落刺激因子(mGM-CSF)和白介素4(mlL-4)联合方案体外诱导小鼠骨髓来源的树突状细胞(Dendritic Cell，DC)，并利用脂多糖(LPS)刺激树突状细胞的成熟。处理组分别于树突状细胞诱导培养的第0天和第5天添加终浓度为0.5mg／ml氧化苦参碱(OMT)，并于培养第7天收集细胞。实验分组为A组：DC；B组：DC+OMT(DO)；C组：DC+OMT(D5)；D组：DC+LPS；E组：DC+OMT(DO)+LPS；F组：DC+OMT(D5)+LPS；G组：T淋巴细胞对照组。采用流式细胞术检测DC表面分子CD40的表达；3H-TdR掺入细胞增殖反应检测氧化苦参碱对树突状细胞的细胞毒作用和混合淋巴细胞反应(MLR)中树突状细胞对T淋巴细胞的刺激能力；采用酶联免疫吸附试验法(ELISA)检测MLR上清中IFN-γ的分泌水平。【结果】1.3H-TdR细胞掺入试验检测氧化苦参碱对小鼠树突状细胞存活的抑制作用。结果氧化苦参碱在浓度为0.8mg／ml时即表现出抑制小鼠树突状细胞的作用(P 0.05)，在1.0mg／ml-5.0mg／ml浓度范围内，氧化苦参碱抑制树突状细胞增殖作用呈剂量依赖性，与对照组相比均具有显著性差异(P 0.01)。2．树突状细胞表面标志CD40的表达：①B组DC表面分子CD40的表达[(40.77±1.28)％]较之对照A组[(25.93±7.85)％]显著升高，有统计学意义(p＜0.01)；C组DC表面分子CD40的表达[(26.40±4.85)％]较之对照A组，无统计学意义。②E组DC表面分子CD40的表达[(48.62±2.46)％]较之对照D组[(34.92±5.82)％]升高，有统计学意义(p＜0.05)。3．混合淋巴细胞反应：①各组DC与T细胞以1：5、1：10和1：20比例混合时T细胞的增殖较T细胞对照组(即G组)均明显增高(P0.05)；随混合比例增加，T细胞增殖也增高。②DC：T为1：5时，B组T细胞的增殖[(49094.67±3048.50)cpm]较之A组[(26628.67±4268.55)cpm]升高，有统计学意义(p 0.05)；C组[(27195.00±9607.25)cpm]较之对照A组无统计学差异。③E组T细胞的增殖[(149144.30±7212.92)cpm]较之对照D组[(120832.30±6825.45)cpm]升高，有统计学意义(p 0.05)；F组[(89665.33±7420.14)cpm]较之对照D组无统计学差异。4．细胞因子检测：①各干预组较T细胞对照组上清中IFN-γ[(4.11±2.51)pg／ml]均有增高，有统计学意义(p＜0.05)。②B组上清中IFN-γ[(181.84±33.16)pg／ml]较之对照A组[(100.88±8.51)pg／ml]高，有统计学意义(p＜0.05)，C组[(98.40±31.19)pg／ml]较之对照A组没有统计学意义；③E组上清中IFN-γ[(716.47±5.38)pg／ml]和F组[(718.33±5.88)pg／ml]较之对照D组[(645.91±81.96)pg／ml]无统计学意义。【结论】氧化苦参碱可以上调正常小鼠树突状细胞表面分子CD40的表达，增强了其促T淋巴细胞增殖和分泌IFN-γ的能力，加强树突状细胞的抗原递呈能力，且对树突状细胞的体外成熟有促进作用，但是其进一步的分子机制还有待进一步研究。更多还原

【Abstract】 【Objective】To investigate the effects of Oxymatrine on maturation and functions of murine bone marrow derived dendritic cells in vitro.【Method】Murine myeloid dendritic cells(DC) were generated from bone marrow in vitro using rmGM-CSF and IL-4, DC were matured by LPS for 48h.Bone marrow derived dendritic cells were treated with 0.5mg/ml Oxymatrine at day 0 and 5,respectively.The control was treatd with medium alone.The phenotypes and functions of DCs were determined at day 7.There were 7 groups in this experiment: group A: DC; group B: DC+OMT (D0); group C: DC+OMT (D5); group D: DC+LPS; group E: DC+OMT(D0)+LPS; group F: DC+OMT(D5)+LPS; group G: T cell control.The phenotype of DC was determined by flow cytometry. The inhibition of dendritic cells and the effect of Oxymatrine on immunno-stimulatory capability of DCs was determined by【3H】Thymidine incorporation assay.ELISA was used to measure the consentration of interferon-γ(IFN-γ) in supernatants of cell cultures.【Result】1. The proliferation of dendritic cells was inhibited by 0.8mg/ml oxymatrine.Ranging from 1.0mg/ml to 5.0mg/ml, oxymatrine had the dose - dependent inhibitory effect on the proliferation of dendritic cells, which showed significant difference compared with control group (P 0.05). 2.The expression of CD40 on DCs:①The expression of CD40 on DCs treated with Oxymatrine at day 0 which was group B was higher than the control group A【n=4, (40.77±1.28)% vs (25.93±7.85)% (p<0.001)】;the expression of CD40 on DCs between group C and A had no significantly difference.②The expression of CD40 on DCs treated with Oxymatrine at day 0 and induced further maturation by LPS,as we named group E was higher than the control group D【n=4,(48.62±2.46)% vs (34.92±5.82)% (p<0.05)】.3.Mixed lymphocyte reaction:①The cellular proliferation of splenocytes stimulated by DCs were higher than the T cell control which was group G(p<0.05), whatever the ratioes between DCs and T cells were 1:5, 1: 10 and 1:20.②When DCs and T cells were in the ratio of 1:5,the cellular proliferation of splenpcytes stimulated by group B DCs was higher than the control group A【n=3, (49094.67±3048.50)cpm vs (26628.67±4268.55)cpm (p<0.05)】.There was no signficant difference between group C and A.③As DCs were induced further maturation by LPS, the cellular proliferation of splenpcytes stimulated by group E DCs was higher than the control group D【n=3, (149144.30±7212.92)cpm vs (120832.30±6825.45)cpm (p<0.05)】.4. We detected the concentration of INF-γin supernatants of MLR,when DCs and T cells were in the ratio of 1:5.①The concentration of INF-γin MLR supernatants of group A-F were higher than the T cell control (p<0.05).②The concentration of INF-γin MLR supematants of group B was higher than the control group A【n=3, (181.84±33.16)pg/ml vs (100.88±8.51)pg/ml (p<0.05)】. There was no signficant difference between group C and A.③As DCs were induced further maturation by LPS, The concentration of INF-γin MLR supematants of group E and F were not higher than the control group A.There were no signigficant difference among these three groups..【Conclusion】Oxymatrine can increasing the expression of CD40 on the dendritic cells and the capacity of stimulating lymphocyte proliferation and excretion ofγ-IFN.It can strengthen the dendritic cells functions of presenting antigen.It can promote the mature of dendritic cells induced by LPS. The specific mechanism need deeper study.更多还原