氧化苦参碱对小鼠树突状细胞成熟和功能的影响
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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。
【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.
【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。
【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.
引文
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[10]．陈小松，王国俊，蔡雄，等．氧化苦参碱对乙型肝炎病毒转基因小鼠乙肝抗原表达的影响[J]．第二军医大学学报，1999，20(10)：746
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[12]．李龙江，陈志琼，郑元义，等．氧化苦参碱对人卵巢癌细胞SKOV-3体外活性的影响[J]．实用肿瘤学杂志，2000，15(5)：297
[13]．王兵，王国俊，徐均．氧化苦参碱对肿瘤诱导血管内皮细胞增殖的抑制作用[J]．中华消化杂志，2001，21(9)：550
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[1]．侯阿丽，等．苦参药用综论中医药学报，1994；(3)：48
[2]. Lai JP, He XW, Jiang Y, etal. Preparative separation and determination of matrine from the Chinese medicinal plant Sophora flavescens Ait by molecularly imprinted solid-phase extraction. Anal Bioanal Chem, 2003, 375(2): 264
[3]．雷载权．中药学，第一版，64-65
[4]．王晓红，黄圣凯．苦参碱及氧化苦参碱的药代动力学与药效动力学[J]．中华内科杂志，2001，40(12)：843
[5]．焦霞，沈其昀，王利民，等．氧化苦参碱对哮喘小鼠抗炎作用的研究[J]．上海第二医科大学学报，2002，22(4)：303
[6]．郑萍，牛凤丽，刘文忠，等．氧化苦参碱对葡聚糖硫酸钠诱导大鼠结肠炎的抗炎作用机制研究[J]．中华消化杂志，2003，23(4)：207
[7]．陈福祥，刘晶星，陆德源．苦参总碱体外抗柯萨基B病毒3型作用测定及其机理的初步研究[J]．中华实验和临床病毒学杂志，1995，9(2)：115
[8]．杨志伟，周娅，曹秀琴．苦豆子生物碱体外抗柯萨奇B3病毒的作用[J]．四川中医，2003，21(3)：14
[9]．李继强，陈萦?，曾民德，等．氧化苦参碱抗乙型肝炎病毒的体外实验研究[J]．中华消化杂志，2001，21(9)：550
[10]．陈小松，王国俊，蔡雄，等．氧化苦参碱对乙型肝炎病毒转基因小鼠乙肝抗原表达的影响[J]．第二军医大学学报，1999，20(10)：746
[11]．孔庆志，黄涛，黄冬生，等．苦参素对小鼠移植性S180肉瘤血管形成的抑制作用[J]．中国药师，2003，12(6)：769
[12]．李龙江，陈志琼，郑元义，等．氧化苦参碱对人卵巢癌细胞SKOV-3体外活性的影响[J]．实用肿瘤学杂志，2000，15(5)：297
[13]．王兵，王国俊，徐均．氧化苦参碱对肿瘤诱导血管内皮细胞增殖的抑制作用[J]．中华消化杂志，2001，21(9)：550
[14]．王会贤，章灵华，杜守英，等．氧化苦参碱对淋巴细胞增殖的影响[J]．中草药，1994；25(7)：362—363
[15]．钱玉昆．中药(苦参及枸杞子)对免疫细胞和细胞因子的实验调节[J]．中华微生物学和免疫学杂志，1988；8(5)：312—314
[16]．韩家文，冯晓军，白惠卿，等．氧化苦参碱对小鼠激活的巨噬细胞吞噬功能的影响[J]．北京医科大学学报，1987；19(4)：269—270
[17]．王会贤，章灵华，黄艺，等．氧化苦参碱对LAK细胞活性的影响[J]．免疫学杂志，1994；10(1)：17—19
[18]．黎雪如，戴寿芝，李为，等．苦豆子的7种生物碱对小鼠免疫功能的影响[J]．中草药，1987；18(5)：22—23
[191]．黄秀梅，李波，沈连忠，等．四种苦豆子生物碱对巨噬细胞产生肿瘤坏死因子的影响[J]．中药 药理与临床，2001；17(3)：12—14
[20]．路歧祥，谭焕然，徐锡荣．苦参碱等五种单体生物碱影响免疫功能的探讨[J]．上海免疫学杂志，1987；7(6)：335—337
[21]．魏立民，张兴荣，马述春，等．生长抑素及氧化苦参碱治疗大鼠急性胰腺炎的实验研究[J]．第二军医大学学报，1999；20(9)：633
[22]．徐广伟，满世军，王志生，等．氧化苦参碱对荷瘤小鼠免疫功能的影响[J]．中国肿瘤临床与康复，2001，8(5)：10
[23]．陈曙霞．苦参生物碱的药理研究进展[J]．中成药，2003，25(1)：75-77
[24]．张宝恒，孔祥军，蔡育立．氧化苦参碱抗心率失常作用[J]．北京医科大学学报，1998，20(8)：572
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