植物活性多糖对小鼠树突状细胞和巨噬细胞功能的调节作用
|
摘要
自然界中植物种类多种多样,具有免疫调节作用的药用植物也是数量繁多,如果能将其中起主要作用的有效成分分离出来,研究其理化性质及作用机理,对于植物功能性因子的研究及相应功能食品的开发将具有非常重要的意义。
抗原提呈细胞(antigen-presenting cell,APCs)主要承担对抗原物质的摄取,加工,并将抗原肽呈递给特异性T淋巴细胞,是启动免疫应答的关键因素。树突状细胞(dendritic cells,DCs),是目前已知功能最强的专职APCs,具有诱导初次免疫应答的独特功能,能有效刺激初始型T细胞(na(l|¨)ve T cells)增殖,在机体细胞免疫和体液免疫调控中具有独特的地位;巨噬细胞作为宿主天然免疫防御阵线的重要成员在先天性免疫防御和获得性免疫应答中同样起着不可忽视的作用。
为探讨中药植物的免疫增强药理机制,本研究初步建立了以小鼠骨髓来源DCs为靶细胞的植物化学物免疫活性筛选模型,在细胞水平初步探讨了大粒车前子精制多糖(crude polysaccharide from the seeds of Plantago asiatica L.,PL-PS)、大粒车前子多糖纯品(polysaccharide from the seeds of Plantago asiatica L.,PLP)、毛蕊花苷(acteoside)、异毛蕊花苷(isoacteoside)、茶叶糖蛋白(tea glycoprotein,TGP)、青钱柳多糖(polysaccharides from Cyclocarya paliurus(Batal.)Iljinskaja,CPC)的免疫调节功能。在此基础上,进一步研究了大粒车前子多糖PL-PS及PLP对小鼠骨髓来源DCs的分化、发育、成熟、迁移等方面的影响,并初步探讨了PL-PS对同为APCs的巨噬细胞功能的影响,为车前子产品开发提供理论依据。现将本文主要研究结果归纳如下:
1.探讨了PLP、CPC、PL-PS、TGP、毛蕊花苷和异毛蕊花苷对DCs前体细胞的细胞毒性。采用倒置显微镜观察细胞形态;用MTT法检测对细胞生长的增殖率;流式细胞仪测定细胞的早期凋亡。结果发现经各植物化学物处理后DCs前体细胞生长良好、细胞生存率高,对DCs前体细胞凋亡无明显影响,青钱柳多糖、毛蕊花苷和异毛蕊花苷对小鼠DCs前体细胞具有显著地促进增殖作用,提示青钱柳多糖、毛蕊花苷和异毛蕊花苷对小鼠DCs前体细胞的影响有可能通过直接诱导扩增及增强其细胞生物活性而发挥。实验结果发现本研究所采用的各植物化学物均无细胞毒性,可用于后续实验,并为临床应用及食用安全提供理论依据。
2.建立了DCs为靶细胞的植物化学物活性筛选模型。参照经典文献,结合国际上研究现状,采用手法分离纯化小鼠骨髓细胞,联合rmGM-CSF和rmIL-4诱导分化培养5天后,用MACS免疫磁珠法对细胞进行进一步纯化。通过采用各种形态学方法观察细胞形态,流式细胞仪分析细胞表面分子表达对细胞进行鉴定,结果发现该方法可以获得大量稳定的高纯度DCs,可用于后续植物化学物的免疫活性筛选。
3.应用已建立的以DCs为靶细胞的活性筛选模型,初步探讨了PLP、CPC、PL-PS、TGP、毛蕊花苷和异毛蕊花苷的免疫调节活性。采用倒置显微镜观察,发现DCs经PLP、CPC、PL-PS、TGP诱导后,细胞周围突起增多,细胞体积增大,形态更加典型。经毛蕊花苷和异毛蕊花苷刺激24 h后,与阴性对照组相比,细胞成团数量有所增加。MTT法检测各植物化学物对DCs增殖的影响,发现大部分植物化学物在相应的浓度范围内,对DCs的生长无显著影响;毛蕊花苷和异毛蕊花苷可促进DCs的增殖。流式细胞仪检测各植物化学物对DCs表型和吞噬功能的影响,发现与目前公认具有免疫促进功效并已应用于肿瘤辅助治疗的香菇多糖相似,PLP、PL-PS、毛蕊花苷和异毛蕊花苷在浓度为50μg/mL,TGP和CPC在浓度为100μg/mL时均可增强CD11c阳性细胞表达MHCⅡ分子。并且发现经各植物活性提取物诱导后DCs吞噬荧光标记的葡聚糖的能力下降。研究结果初步发现PLP、CPC、PL-PS、TGP、毛蕊花苷和异毛蕊花苷均具有促进DCs表型及功能成熟的作用,从而为后续进一步实验提供理论依据。
4.探讨了不同剂量大粒车前子多糖PL-PS及PLP对DCs表型及吞噬功能的影响。采用流式细胞分析技术分析不同剂量PL-PS和PLP对DCs表面MHCⅡ、CD80、CD86及CD40表达水平的影响。结果发现PL-PS和PLP均可促进MHCⅡ、CD80、CD86和CD40的表达,在10～200μg/mL浓度范围内,呈剂量依赖关系。且PL-PS、PLP同剂量组相比,在对CD86分子表达上,PL-PS高、中剂量组比同剂量组的PLP强(p＜0.05),在对CD80的影响的表达上,PL-PS低剂量组比同剂量组的PLP强(p＜0.05)。空白组DCs吞噬FITC标记的葡聚糖功能很强,而经不同剂量PL-PS,PLP处理后DCs的吞噬活性均明显下降。再次提示大粒车前子多糖可促进小鼠骨髓来源DCs表型及功能的成熟,并且这一促进功能在10～200μg/mL浓度范围内,呈剂量依赖关系。
5.探讨了不同剂量大粒车前子多糖PL-PS及PLP对DCs分泌功能的影响。采用Griess法检测各组DCs分泌NO的量;双抗体夹心法酶联免疫吸附实验(ELISA)检测细胞培养液上清中细胞因子IL-12p70、IL-10、IL-1β及趋化因子RANTES的含量。结果发现,PL-PS及PLP能够诱导未成熟DCs分泌Th1型细胞因子IL-12p70和IL-1β,对Th2型细胞因子IL-10则具有显著抑制作用(p＜0.05),并且这种刺激作用具有剂量依赖性,与同剂量的PLP相比,25μg/mL PL-PS可显著降低IL-10的产生(p＜0.05)。PL-PS及PLP能够刺激未成熟DCs分泌RANTES-Th1型趋化因子,而且这种促进作用在PL-PS浓度为25μg/mL时最显著(p＜0.05)。由此可见,PL-PS及PLP可以通过调节DCs分泌不同类型的细胞因子及趋化因子,诱导机体偏向Th1型细胞免疫应答,以及促进机体天然免疫向获得性免疫过渡。
6.探讨了不同剂量大粒车前子多糖PL-PS及PLP对DCs刺激T细胞功能的影响。采用MTT法检测PL-PS及PLP对DCs刺激OVA未致敏或致敏淋巴细胞增殖的影响,发现PL-PS和PLP均可增强树突状细胞的刺激未致敏和致敏淋巴细胞增殖的能力,说明经PL-PS或PLP刺激后,树突状细胞诱导免疫应答及抗原提呈功能均增强。提示在适宜的T:DCs比时,PL-PS与PLP的作用一致,均能促进DCs对T细胞增殖的刺激作用。
7.探讨了不同剂量大粒车前子多糖PL-PS及PLP对DCs趋化功能的影响。采用半定量PCR法检测了DCs表面趋化因子受体CCR7 mRNA的表达水平。结果发现PL-PS或PLP均可影响DCs表面趋化因子受体CCR7的表达。其中,与阴性对照相比,PL-PS在浓度为100μg/mL,PLP在浓度为50μg/mL和100μg/mL时,可显著提高DCs表面CCR7的表达(p＜0.01)。可以推测PLP或PL-PS对DCs的促成熟作用可能与趋化因子受体的表达有关。
8.初步探讨了大粒车前子多糖对DCs抗小鼠SP2/0骨髓瘤功能的影响。选取小鼠骨髓瘤细胞制备冻融抗原,使小鼠骨髓来源DCs负载SP2/0细胞冻融抗原,致敏T淋巴细胞,产生CTL,诱导出的T淋巴细胞对SP2/0细胞有明显CTL活性,而PL-PS及PLP的干预可促进这一功能的提高。所以大粒车前子多糖抗肿瘤机制与促进DCs抗原呈递能力有密切的关系,可提高机体对肿瘤细胞的特异性主动免疫功能。
9.探讨了大粒车前子多糖对小鼠巨噬细胞功能的影响。采用倒置显微镜、MTT法、Griess法、流式细胞术及ELISA法探讨了大粒车前子多糖对RAW264.7细胞株及小鼠腹腔巨噬细胞的影响。结果发现:PL-PS能刺激RAW264.7细胞株和小鼠腹腔巨噬细胞生成NO;促进RAW264.7细胞表面CD80、CD86、CD40的表达及TNF-α、IL-1β的分泌,并能促进腹腔巨噬细胞IL-10的分泌。提示大粒车前子多糖的免疫调节活性也与其对巨噬细胞的作用相关。
上述结果提示:植物化学物的免疫调节可通过建立的以小鼠骨髓来源DCs为靶细胞的模型来进行初步的筛选和探讨。大粒车前子多糖PL-PS及PLP可通过促进DCs表面MHCⅡ类分子,共刺激分子的表达及Th1型细胞因子的分泌,刺激T淋巴细胞的增殖,诱导Th1型免疫应答并增强CTL对肿瘤细胞的特异性杀伤活力。并通过促进DCs表面趋化因子受体CCR7 mRNA的表达,促进DCs的移行和发育成熟。说明PL-PS及PLP均作用于DCs,通过对DCs移行、成熟、功能的上调作用而促进免疫应答的启动,增强机体的免疫功能。另外,PL-PS也可通过对巨噬细胞的作用来调节机体免疫应答。
There are numerous plants in the nature,among which many might be used as immunomodulator agents.It is significant to separate the effective components and study on their biological properties,which will provide scientific functions for their further developments as active ingredients in functional food,or new medicine.
Antigen-presenting cells(APCs) whose primary function is to capture,process, and present antigens toward T lymphocytes plays an important role in initiating T cell responses against microbial pathogens and tumors.Dendritic cells(DCs) are the most potent professional APCs with distinct abilities to stimulate na(l|¨)ve T lymphocytes and initiate primary immune responses.Given their central role in controlling immunity, DCs are targets for many situations involving anti-infection or anti-tumor immunologic response.
In order to illustrate the possible pharmacological mechanism of those medical functions of Chinese medicines from the aspect of immunology,a screening model of immunological activity of phytochemicals by using DCs as the target cells was established.Effects of CPC,TGP,PL-PS,PLP,isoactoside and acteoside on the phenotypic and functional maturation of DCs were tested by using this screening model.Based on the preliminary results,the effects of PL-PS or PLP on the differentiation,development,maturation,and immigration of DCs were studied.In addition,the activation of PL-PS on macrophage which also belongs to APCs was investigated.The purpose of this study was to make a preliminary investigation of the immunomodulation mechanism of the polysaccharides and to provide theoretical foundation for the exploration of products made from the seeds of Plantago asiatica L..Moreover.the screening model established in our study will provide a new approach for the bioactivity-screening studies.The main research results obtained in this dissertation are concluded as follows:
1.Investigated the cytotoxity of PLP.CPC,PL-PS,TGP,acteoside and isoacteoside on DCs precursor.We observed the effects of these extracts on cellular morphology with inverted microscope,used MTT method to determine the inhibitory rate of these extracts on cell growth,and applied flow cytometry to determine apoptosis.We found that a series of concentrations of these extracts had neither reliable effect on cell morphology,nor significant inhibition on cell growth and proliferation.The cytotoxicity gradation of various concentrations of these extracts ranged from 0 to 1 degree.The same results were discovered by apoptosis analyses. Thus,we came to the conclusion that in a certain scope of concentration,the extracts we used have no cytotoxity on DCs precursor,and can be used in the selection-of immunopotentiating agent.
2.The screening model of immunological activity by using DCs as target cells was established.Based on the classical literature and the current study,we induced the murine bone marrow to immature dendritic cells by rmGM-CSF and rmIL-4.On day 5,the cells were collected,and CD11c~+ cells were isolated by MACS.The morphology were observed by using inverted microscope,scanning electron microscope and transmission electron microscope.While the expression level of surface molecular on DCs were tested by flow cytometry.The results showed that high purity of DCs can be gained,and the method was stabiliy,which could used for bioactivity-screening studies.
3.Investigate the immunological activities of CPC.TGP,PL-PS,PLP, isoactoside and acteoside by using the bioactivity-screening model.We observed the DCs after being stimulated by these extracts with inverted microscopic,and finded that these colonies were covered with many sheet-like processes typical of DCs.The effects of PLP,CPC,PL-PS,TGP,acteoside and isoacteoside on proliferation of dendritic cells in vitro were studied by using MTT assay and the results showed that CPC,TGP.PL-PS and PLP have no effects on the proliferation of DCs in certain concertrations,whereas isoactoside and acteoside could significantly stimulate the proliferation of DCs.Flow cytometer showed that like lentinan,all these extracts origin from plane medicine could enhance the expression levels of MHCⅡon DCs surfaces which gated on CD11c~+ DCs,and decrease the uptake dextran by DCs compared with unstimulated DCs.
4.Investigated the effects of PL-PS or PLP on the phenotype and phagocytosis on DCs.Flow cytometroy were used to analyze the expression level of MHC classⅡ. co-stimulate molecule CD80,CD86 and CD40 on DCs.We found that both PL-PS and PLP could enhance the expression level of MHCⅡ,CD80,CD86 and CD40.At the four doses(from 10μg/mL to 200μg/mL) tested,there was a concentration-dependent relationship.The effects of PL-PS on the expresision of CD86 on DCs were higher than the same dose of PLP at the medial and high concentration(p＜0.05).Untreated DCs showed high phagocytosis to FITC-dextran, and after being treated with PL-PS or PLP,the phagocytosis were significantly decreased(p＜0.05),which showed a concentration-dependent relationship at the four doses(from 10μg/mL to 200μg/mL) tested.
5.Investigated the effects of PL-PS or PLP on the secretion activity of DCs.The level of NO was detected by NO analyzing Kit.The level of mouse IL-12 p70,IL-10, IL-1βand chemotatic factor RANTES in culture supernatants was determined by sandwich enzyme-linked immunosorbent assay.We found that both PL-PS and PLP could enhance the secretion of Th-1 related cytokine like IL-12 p70 and IL-1β,and this effect was dose-depended.With the IL-10 secretion of DCs,both PL-PS and PLP could inhibit significantly the IL-10 secretion(p＜0.05) and the effect of PL-PS is more significant than the same concentration of PLP(p＜0.01).The secretion of RANTES of DCs was also elevated after being treated with PL-PS or PLP.These results showed that both PL-PS and PLP could induce cellullar immunologic response by affecting the cytokine secretion of DCs.
6.Investigated the effects of PL-PS or PLP on the proliferation of T cell stimulated by DCs.Antigen presenting ability to allogeneically naive or syngeneically primed T lymphocytes was examined by the lymphocyte proliferation of mixed lymphocyte reaction(MLR).The result demonstrated that both PL-PS and PLP could increase antigen presenting ability of DCs to allogeneically na(l|¨)ve or syngeneically primed T lymphocytes.Both PL-PS and PLP at high concentration play stimulating effects on the proliferation of T cell when the ratio between T cell and DCs was 10:1.
7.Investigated the effects of PL-PS or PLP on the expression level of CCR7 mRNA on DCs.Through seim-quantitation PCR method,the expression of chemotactic receptor CCR7 on DCs surface was analyzed to clarify the molecular mechanism that PL-PS or PLP changes chemotactic activity of DCs.Results show that PL-PS at the concentration of 100μg/mL,PLP at the concentration of 50 and 100μg/mL could increse the expression of CCR7,when compared to the untreated group (p＜0.01).The results showed that both PL-PS and PLP have influence on chemotactic factor receptor of DCs surface.
8.Investigate the effect of PL-PS or PLP on cytotoxicity of specific cytotoxic T-lymphocytes(CTL) induced by DCs.Cultured murine DCs were pulsed with SP2/0 tumor cell lysates and co-incubated with PL-PS or PLP.SP2/0 specific CTL were induced by spleen lymphocytes stimulated with DCs.The results showed that both PL-PS and PLP promoted LDH activities released into culture supernatants.Thus,we came to the conclusion that both PL-PS and PLP could promote the cytotoxicity of specific CTL induced by DCs which pulsed with SP2/0 tumor antigen durning the stage of antigen presentation.
9.The immunomodulatory activities of PL-PS were examined on RAW 264.7 and murine peritoneal macrophage.By using MTT method,Griess method,flow cytometry and ELISA method,we found that PL-PS could activated RAW264.7 cells to produce cytokines such as tumor necrosis factor(TNF)-α,IL-1β,and nitric oxide (NO)dose dependently.It induced the expression of co-stimulatory molecules such as B7-1,B7-2 and CD40 on RAW264.7 cells.PL-PS could also activated murine peritoneal macrophage s to produce NO and IL-10.These results show that PL-PS possess potent immunomodulatory activity on macrophage lineage cells.
The results demonstrated that both PL-PS and PLP could improve the proliferation of T cell and enhance the specific cytotoxity activity of CTL by improving the expression of adhesionand co-stimulating moleculars on the surface of DCs and secretion of Th1 related cytokines.By elevating the exprssion level of CCR7 on DCs,both PL-PS and PLP can enhance the migration of DCs.In addition. PL-PS could enhance the activation of macrophage lineage cells.These results may provide experimental basis of pharmacodynamics for clinical application of PL-PS and PLP.
抗原提呈细胞(antigen-presenting cell,APCs)主要承担对抗原物质的摄取,加工,并将抗原肽呈递给特异性T淋巴细胞,是启动免疫应答的关键因素。树突状细胞(dendritic cells,DCs),是目前已知功能最强的专职APCs,具有诱导初次免疫应答的独特功能,能有效刺激初始型T细胞(na(l|¨)ve T cells)增殖,在机体细胞免疫和体液免疫调控中具有独特的地位;巨噬细胞作为宿主天然免疫防御阵线的重要成员在先天性免疫防御和获得性免疫应答中同样起着不可忽视的作用。
为探讨中药植物的免疫增强药理机制,本研究初步建立了以小鼠骨髓来源DCs为靶细胞的植物化学物免疫活性筛选模型,在细胞水平初步探讨了大粒车前子精制多糖(crude polysaccharide from the seeds of Plantago asiatica L.,PL-PS)、大粒车前子多糖纯品(polysaccharide from the seeds of Plantago asiatica L.,PLP)、毛蕊花苷(acteoside)、异毛蕊花苷(isoacteoside)、茶叶糖蛋白(tea glycoprotein,TGP)、青钱柳多糖(polysaccharides from Cyclocarya paliurus(Batal.)Iljinskaja,CPC)的免疫调节功能。在此基础上,进一步研究了大粒车前子多糖PL-PS及PLP对小鼠骨髓来源DCs的分化、发育、成熟、迁移等方面的影响,并初步探讨了PL-PS对同为APCs的巨噬细胞功能的影响,为车前子产品开发提供理论依据。现将本文主要研究结果归纳如下:
1.探讨了PLP、CPC、PL-PS、TGP、毛蕊花苷和异毛蕊花苷对DCs前体细胞的细胞毒性。采用倒置显微镜观察细胞形态;用MTT法检测对细胞生长的增殖率;流式细胞仪测定细胞的早期凋亡。结果发现经各植物化学物处理后DCs前体细胞生长良好、细胞生存率高,对DCs前体细胞凋亡无明显影响,青钱柳多糖、毛蕊花苷和异毛蕊花苷对小鼠DCs前体细胞具有显著地促进增殖作用,提示青钱柳多糖、毛蕊花苷和异毛蕊花苷对小鼠DCs前体细胞的影响有可能通过直接诱导扩增及增强其细胞生物活性而发挥。实验结果发现本研究所采用的各植物化学物均无细胞毒性,可用于后续实验,并为临床应用及食用安全提供理论依据。
2.建立了DCs为靶细胞的植物化学物活性筛选模型。参照经典文献,结合国际上研究现状,采用手法分离纯化小鼠骨髓细胞,联合rmGM-CSF和rmIL-4诱导分化培养5天后,用MACS免疫磁珠法对细胞进行进一步纯化。通过采用各种形态学方法观察细胞形态,流式细胞仪分析细胞表面分子表达对细胞进行鉴定,结果发现该方法可以获得大量稳定的高纯度DCs,可用于后续植物化学物的免疫活性筛选。
3.应用已建立的以DCs为靶细胞的活性筛选模型,初步探讨了PLP、CPC、PL-PS、TGP、毛蕊花苷和异毛蕊花苷的免疫调节活性。采用倒置显微镜观察,发现DCs经PLP、CPC、PL-PS、TGP诱导后,细胞周围突起增多,细胞体积增大,形态更加典型。经毛蕊花苷和异毛蕊花苷刺激24 h后,与阴性对照组相比,细胞成团数量有所增加。MTT法检测各植物化学物对DCs增殖的影响,发现大部分植物化学物在相应的浓度范围内,对DCs的生长无显著影响;毛蕊花苷和异毛蕊花苷可促进DCs的增殖。流式细胞仪检测各植物化学物对DCs表型和吞噬功能的影响,发现与目前公认具有免疫促进功效并已应用于肿瘤辅助治疗的香菇多糖相似,PLP、PL-PS、毛蕊花苷和异毛蕊花苷在浓度为50μg/mL,TGP和CPC在浓度为100μg/mL时均可增强CD11c阳性细胞表达MHCⅡ分子。并且发现经各植物活性提取物诱导后DCs吞噬荧光标记的葡聚糖的能力下降。研究结果初步发现PLP、CPC、PL-PS、TGP、毛蕊花苷和异毛蕊花苷均具有促进DCs表型及功能成熟的作用,从而为后续进一步实验提供理论依据。
4.探讨了不同剂量大粒车前子多糖PL-PS及PLP对DCs表型及吞噬功能的影响。采用流式细胞分析技术分析不同剂量PL-PS和PLP对DCs表面MHCⅡ、CD80、CD86及CD40表达水平的影响。结果发现PL-PS和PLP均可促进MHCⅡ、CD80、CD86和CD40的表达,在10～200μg/mL浓度范围内,呈剂量依赖关系。且PL-PS、PLP同剂量组相比,在对CD86分子表达上,PL-PS高、中剂量组比同剂量组的PLP强(p＜0.05),在对CD80的影响的表达上,PL-PS低剂量组比同剂量组的PLP强(p＜0.05)。空白组DCs吞噬FITC标记的葡聚糖功能很强,而经不同剂量PL-PS,PLP处理后DCs的吞噬活性均明显下降。再次提示大粒车前子多糖可促进小鼠骨髓来源DCs表型及功能的成熟,并且这一促进功能在10～200μg/mL浓度范围内,呈剂量依赖关系。
5.探讨了不同剂量大粒车前子多糖PL-PS及PLP对DCs分泌功能的影响。采用Griess法检测各组DCs分泌NO的量;双抗体夹心法酶联免疫吸附实验(ELISA)检测细胞培养液上清中细胞因子IL-12p70、IL-10、IL-1β及趋化因子RANTES的含量。结果发现,PL-PS及PLP能够诱导未成熟DCs分泌Th1型细胞因子IL-12p70和IL-1β,对Th2型细胞因子IL-10则具有显著抑制作用(p＜0.05),并且这种刺激作用具有剂量依赖性,与同剂量的PLP相比,25μg/mL PL-PS可显著降低IL-10的产生(p＜0.05)。PL-PS及PLP能够刺激未成熟DCs分泌RANTES-Th1型趋化因子,而且这种促进作用在PL-PS浓度为25μg/mL时最显著(p＜0.05)。由此可见,PL-PS及PLP可以通过调节DCs分泌不同类型的细胞因子及趋化因子,诱导机体偏向Th1型细胞免疫应答,以及促进机体天然免疫向获得性免疫过渡。
6.探讨了不同剂量大粒车前子多糖PL-PS及PLP对DCs刺激T细胞功能的影响。采用MTT法检测PL-PS及PLP对DCs刺激OVA未致敏或致敏淋巴细胞增殖的影响,发现PL-PS和PLP均可增强树突状细胞的刺激未致敏和致敏淋巴细胞增殖的能力,说明经PL-PS或PLP刺激后,树突状细胞诱导免疫应答及抗原提呈功能均增强。提示在适宜的T:DCs比时,PL-PS与PLP的作用一致,均能促进DCs对T细胞增殖的刺激作用。
7.探讨了不同剂量大粒车前子多糖PL-PS及PLP对DCs趋化功能的影响。采用半定量PCR法检测了DCs表面趋化因子受体CCR7 mRNA的表达水平。结果发现PL-PS或PLP均可影响DCs表面趋化因子受体CCR7的表达。其中,与阴性对照相比,PL-PS在浓度为100μg/mL,PLP在浓度为50μg/mL和100μg/mL时,可显著提高DCs表面CCR7的表达(p＜0.01)。可以推测PLP或PL-PS对DCs的促成熟作用可能与趋化因子受体的表达有关。
8.初步探讨了大粒车前子多糖对DCs抗小鼠SP2/0骨髓瘤功能的影响。选取小鼠骨髓瘤细胞制备冻融抗原,使小鼠骨髓来源DCs负载SP2/0细胞冻融抗原,致敏T淋巴细胞,产生CTL,诱导出的T淋巴细胞对SP2/0细胞有明显CTL活性,而PL-PS及PLP的干预可促进这一功能的提高。所以大粒车前子多糖抗肿瘤机制与促进DCs抗原呈递能力有密切的关系,可提高机体对肿瘤细胞的特异性主动免疫功能。
9.探讨了大粒车前子多糖对小鼠巨噬细胞功能的影响。采用倒置显微镜、MTT法、Griess法、流式细胞术及ELISA法探讨了大粒车前子多糖对RAW264.7细胞株及小鼠腹腔巨噬细胞的影响。结果发现:PL-PS能刺激RAW264.7细胞株和小鼠腹腔巨噬细胞生成NO;促进RAW264.7细胞表面CD80、CD86、CD40的表达及TNF-α、IL-1β的分泌,并能促进腹腔巨噬细胞IL-10的分泌。提示大粒车前子多糖的免疫调节活性也与其对巨噬细胞的作用相关。
上述结果提示:植物化学物的免疫调节可通过建立的以小鼠骨髓来源DCs为靶细胞的模型来进行初步的筛选和探讨。大粒车前子多糖PL-PS及PLP可通过促进DCs表面MHCⅡ类分子,共刺激分子的表达及Th1型细胞因子的分泌,刺激T淋巴细胞的增殖,诱导Th1型免疫应答并增强CTL对肿瘤细胞的特异性杀伤活力。并通过促进DCs表面趋化因子受体CCR7 mRNA的表达,促进DCs的移行和发育成熟。说明PL-PS及PLP均作用于DCs,通过对DCs移行、成熟、功能的上调作用而促进免疫应答的启动,增强机体的免疫功能。另外,PL-PS也可通过对巨噬细胞的作用来调节机体免疫应答。
There are numerous plants in the nature,among which many might be used as immunomodulator agents.It is significant to separate the effective components and study on their biological properties,which will provide scientific functions for their further developments as active ingredients in functional food,or new medicine.
Antigen-presenting cells(APCs) whose primary function is to capture,process, and present antigens toward T lymphocytes plays an important role in initiating T cell responses against microbial pathogens and tumors.Dendritic cells(DCs) are the most potent professional APCs with distinct abilities to stimulate na(l|¨)ve T lymphocytes and initiate primary immune responses.Given their central role in controlling immunity, DCs are targets for many situations involving anti-infection or anti-tumor immunologic response.
In order to illustrate the possible pharmacological mechanism of those medical functions of Chinese medicines from the aspect of immunology,a screening model of immunological activity of phytochemicals by using DCs as the target cells was established.Effects of CPC,TGP,PL-PS,PLP,isoactoside and acteoside on the phenotypic and functional maturation of DCs were tested by using this screening model.Based on the preliminary results,the effects of PL-PS or PLP on the differentiation,development,maturation,and immigration of DCs were studied.In addition,the activation of PL-PS on macrophage which also belongs to APCs was investigated.The purpose of this study was to make a preliminary investigation of the immunomodulation mechanism of the polysaccharides and to provide theoretical foundation for the exploration of products made from the seeds of Plantago asiatica L..Moreover.the screening model established in our study will provide a new approach for the bioactivity-screening studies.The main research results obtained in this dissertation are concluded as follows:
1.Investigated the cytotoxity of PLP.CPC,PL-PS,TGP,acteoside and isoacteoside on DCs precursor.We observed the effects of these extracts on cellular morphology with inverted microscope,used MTT method to determine the inhibitory rate of these extracts on cell growth,and applied flow cytometry to determine apoptosis.We found that a series of concentrations of these extracts had neither reliable effect on cell morphology,nor significant inhibition on cell growth and proliferation.The cytotoxicity gradation of various concentrations of these extracts ranged from 0 to 1 degree.The same results were discovered by apoptosis analyses. Thus,we came to the conclusion that in a certain scope of concentration,the extracts we used have no cytotoxity on DCs precursor,and can be used in the selection-of immunopotentiating agent.
2.The screening model of immunological activity by using DCs as target cells was established.Based on the classical literature and the current study,we induced the murine bone marrow to immature dendritic cells by rmGM-CSF and rmIL-4.On day 5,the cells were collected,and CD11c~+ cells were isolated by MACS.The morphology were observed by using inverted microscope,scanning electron microscope and transmission electron microscope.While the expression level of surface molecular on DCs were tested by flow cytometry.The results showed that high purity of DCs can be gained,and the method was stabiliy,which could used for bioactivity-screening studies.
3.Investigate the immunological activities of CPC.TGP,PL-PS,PLP, isoactoside and acteoside by using the bioactivity-screening model.We observed the DCs after being stimulated by these extracts with inverted microscopic,and finded that these colonies were covered with many sheet-like processes typical of DCs.The effects of PLP,CPC,PL-PS,TGP,acteoside and isoacteoside on proliferation of dendritic cells in vitro were studied by using MTT assay and the results showed that CPC,TGP.PL-PS and PLP have no effects on the proliferation of DCs in certain concertrations,whereas isoactoside and acteoside could significantly stimulate the proliferation of DCs.Flow cytometer showed that like lentinan,all these extracts origin from plane medicine could enhance the expression levels of MHCⅡon DCs surfaces which gated on CD11c~+ DCs,and decrease the uptake dextran by DCs compared with unstimulated DCs.
4.Investigated the effects of PL-PS or PLP on the phenotype and phagocytosis on DCs.Flow cytometroy were used to analyze the expression level of MHC classⅡ. co-stimulate molecule CD80,CD86 and CD40 on DCs.We found that both PL-PS and PLP could enhance the expression level of MHCⅡ,CD80,CD86 and CD40.At the four doses(from 10μg/mL to 200μg/mL) tested,there was a concentration-dependent relationship.The effects of PL-PS on the expresision of CD86 on DCs were higher than the same dose of PLP at the medial and high concentration(p＜0.05).Untreated DCs showed high phagocytosis to FITC-dextran, and after being treated with PL-PS or PLP,the phagocytosis were significantly decreased(p＜0.05),which showed a concentration-dependent relationship at the four doses(from 10μg/mL to 200μg/mL) tested.
5.Investigated the effects of PL-PS or PLP on the secretion activity of DCs.The level of NO was detected by NO analyzing Kit.The level of mouse IL-12 p70,IL-10, IL-1βand chemotatic factor RANTES in culture supernatants was determined by sandwich enzyme-linked immunosorbent assay.We found that both PL-PS and PLP could enhance the secretion of Th-1 related cytokine like IL-12 p70 and IL-1β,and this effect was dose-depended.With the IL-10 secretion of DCs,both PL-PS and PLP could inhibit significantly the IL-10 secretion(p＜0.05) and the effect of PL-PS is more significant than the same concentration of PLP(p＜0.01).The secretion of RANTES of DCs was also elevated after being treated with PL-PS or PLP.These results showed that both PL-PS and PLP could induce cellullar immunologic response by affecting the cytokine secretion of DCs.
6.Investigated the effects of PL-PS or PLP on the proliferation of T cell stimulated by DCs.Antigen presenting ability to allogeneically naive or syngeneically primed T lymphocytes was examined by the lymphocyte proliferation of mixed lymphocyte reaction(MLR).The result demonstrated that both PL-PS and PLP could increase antigen presenting ability of DCs to allogeneically na(l|¨)ve or syngeneically primed T lymphocytes.Both PL-PS and PLP at high concentration play stimulating effects on the proliferation of T cell when the ratio between T cell and DCs was 10:1.
7.Investigated the effects of PL-PS or PLP on the expression level of CCR7 mRNA on DCs.Through seim-quantitation PCR method,the expression of chemotactic receptor CCR7 on DCs surface was analyzed to clarify the molecular mechanism that PL-PS or PLP changes chemotactic activity of DCs.Results show that PL-PS at the concentration of 100μg/mL,PLP at the concentration of 50 and 100μg/mL could increse the expression of CCR7,when compared to the untreated group (p＜0.01).The results showed that both PL-PS and PLP have influence on chemotactic factor receptor of DCs surface.
8.Investigate the effect of PL-PS or PLP on cytotoxicity of specific cytotoxic T-lymphocytes(CTL) induced by DCs.Cultured murine DCs were pulsed with SP2/0 tumor cell lysates and co-incubated with PL-PS or PLP.SP2/0 specific CTL were induced by spleen lymphocytes stimulated with DCs.The results showed that both PL-PS and PLP promoted LDH activities released into culture supernatants.Thus,we came to the conclusion that both PL-PS and PLP could promote the cytotoxicity of specific CTL induced by DCs which pulsed with SP2/0 tumor antigen durning the stage of antigen presentation.
9.The immunomodulatory activities of PL-PS were examined on RAW 264.7 and murine peritoneal macrophage.By using MTT method,Griess method,flow cytometry and ELISA method,we found that PL-PS could activated RAW264.7 cells to produce cytokines such as tumor necrosis factor(TNF)-α,IL-1β,and nitric oxide (NO)dose dependently.It induced the expression of co-stimulatory molecules such as B7-1,B7-2 and CD40 on RAW264.7 cells.PL-PS could also activated murine peritoneal macrophage s to produce NO and IL-10.These results show that PL-PS possess potent immunomodulatory activity on macrophage lineage cells.
The results demonstrated that both PL-PS and PLP could improve the proliferation of T cell and enhance the specific cytotoxity activity of CTL by improving the expression of adhesionand co-stimulating moleculars on the surface of DCs and secretion of Th1 related cytokines.By elevating the exprssion level of CCR7 on DCs,both PL-PS and PLP can enhance the migration of DCs.In addition. PL-PS could enhance the activation of macrophage lineage cells.These results may provide experimental basis of pharmacodynamics for clinical application of PL-PS and PLP.
引文
[1]黄亚东,程树军.树突状细胞提取与鉴定方法的研究进展[J].华南国防医学杂志,2008,22(4):74-77.
[2]Dannull J,Cerny T,Ackermann DK,et al.Current status of dendritic cell-based tumor vaccination[J].Onkologie,2000,23(6):544-551.
[3]Hows J M,Marsh J C,Bradley B A,et al.Human cord blood:a source of transplantable stem cells[J].Bone Marrow Transplant,1992,9(Suppl 1):105-108.
[4]Oki M,Ando K,Hagihara M,et al.Efficient lentiviral transduction of human cord blood CD34~+ cells followed by their expansion and differentiation into dendritic cells[J].Experimental Hematology,2001,29(10):1210-1217.
[5]Klinkert W E,La Baddie J H,Bowers E.Accessory and stimulating properties of dendritic cells and macrophages isolated from various rat tissues[J].Journal of Experimental Medicine,1982,156(1):1-19.
[6]许亚明,迟诏丞,任明,等.树突状细胞的研究进展[J].吉林医学,2008,29(13):1127-1130.
[7]Rao M S,Mattson M P.Stem cells and aging:expanding the possibilities[J].Mechanisms of Ageing and Development,2001,122(7):713-734.
[8]侯治富,郭楠,高申,等.人外周血树突状细胞的诱导与鉴定[J].吉林大学学报(医学版),2005,31(5):657-659.
[9]Pea-Cruz V,Ito S,Oukka M,et al.Extraction of human Langerhans cells:a method for isolation of epidermis-resident dendritic cells[J].Journal of Immunological Methods,2001,255(1-2):83-91.
[10]郑茂荣,范青源,谢勇,等.Percoll密度梯度离心技术分离纯化豚鼠表皮Langerhans细胞[J].中华皮肤科杂志,1995,28(5):296-298.
[11]舒赛男,魏来,方峰,等.免疫磁珠分选系统在分离大鼠骨髓干细胞群中的应用[J].标记免疫分析与临床,2006,13(1):35-37.
[12]Alessandro A,Stephanie H,Michael F,et al.F.100.GM-CSF induced maturation of dendritic cells is associated with GSK-3beta Inhibition[J].Clinical Immunology,2008,127(1):76.
[13]Markowicz S,Engleman E G.Granulocyte-macrophage colony-stimulating factor promotes differentiation and survival of human perpheral blood dendritic cells in vivo[J].Journal of Clinical Investigation[J],1990,85:955-61
[14]熊菲.影响树突状细胞分化成熟因素的研究进展[J].国外医学免疫学分册,2005,28(2)107-111.
[15]Santini S M,Di Pucchio T,Lapenta C.et al.A new type Ⅰ IFN-mediated pathway for the rapid differentiation of monocytes into highly active dendritic cells[J].Stem Cells,2003,21(3):357-362.
[16] Reid C D. The dendritic cell lineage in haemopoiesis [J]. British Journal of Haematology,1997, 96(2): 217-223.
[17] Lutz M B, Schnare M, Menges M, et al. Differential functions of IL-4 receptor types I and II for dendritic cell maturation and IL-12 production and their dependency on GM-CSF [J]. Journal of Immunology, 2002, 169(7): 3574-3580.
[18] Jacobsen S E, Ruscetti F W, Dubois C M, et al. Tumor necrosis factor a directly and indirectly regulates hematopoietic progenitor cell proliferation: role of colony-stimulating factor receptor modulation [J]. Journal of Experimental Medicine, 1992, 175(6): 1795—1772.
[19] Chen X, Regn S, Raffegerst S, et al. Interferon alpha in combination with GM-CSF induces the differentiation of leukaemic antigen-presenting cells that have the capacity to stimulate a specific anti-leukaemic cytotoxic T-cell response from patients with chronic myeloid leukemia [J].British Journal of Haematology, 2000, 111(2): 596—607.
[20] Urn H D, Cho Y H, Kimdo K, et al. TNF-alpha suppresses dendritic cell death and the production of reactive oxygen intermediates induced by plasma withdrawal [J]. Experimental Dermatology, 2004, 13(5): 282-288.
[21]裴雪涛,王立生,徐黎,等.CD34+造血祖细胞的定向诱导分化研究[J].中华血液学杂志,1998,19(6):289-293．
[22] Piacibello W, Sanavio F, Garetto L, et al. Differential growth factor requirement of primitives cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation [J]. Leukemia, 1998, 12(5): 718—721.
[23] Maraskovsky E, Daro E, Roux E, et al. In vivo generation of human dendritic cell subsets by Flt3ligand [J]. Blood, 2000, 96(3): 878-884.
[24] Riedl E, Strobl H, Majdic O, et al. TGF-beta1 promotes in vitro generation of dendritic cells by protecting progenitor cells from apoptosis [J]. Journal of Immunology, 1997, 158(4): 1591 —1597.
[25] Allavena P, Piemonti L, Longoni D, et al. IL-10 prevents the differentiation of monocytes to dendritic cells but promotes their maturation to macrophages [J]. European Journal of Immunology, 1998, 28(1): 359-369.
[26] Moore K W, de Waal Malefyt R, Coffman RL, et al. Interleukin-10 and the interleukin-10 receptor [J]. Annual Review of Immunology. 2001. 19: 683—765.
[27] Shortman K, Caux C. dendritic cells development:multiple pathways to nature's adjuvants [J].Stem Cells, 1997, 15(6): 409-419.
[28] Thomas R, Davis L, Lipsley P E. Comparative accessory cell of human peripheral blood DC and monocytes [J]. Journal of Immunology. 1993, 151(12): 6840-6852.
[29] Banchereau J, Steinman R M. Dendritic cells and the control of immunity [J]. Nature, 1998,392:245-252.
[30] Hart D N J. Dendritic cells: unique leukocyte populations which control the primary immune response [J]. Blood, 1997, 90(9): 3245-3287.
[31] Brocker T, Riedinger M. Karjalainen K. Targeted expression of majorhisto compatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo [J]. Journal of Experimental Medicine, 1997, 185(3):541-550.
[32] Brocker T. Survival of mature CD4 T lymphocytes is dependent on major histocompatibility comphex class II-expressing dendritic cells [J]. Journal of Experimental Medicine, 1997, 186:1223-1232.
[33] Fayette J, Dubois B, Vandenabeele S. et al. Human dendritic cells skew isotype switching of CD40 activated naive B cells towards IgA 1 and IgA2 [J]. Journal of Experimental Medicine,1997,185:1909-1918.
[34] Dubois B, Vanbervliet B, Fayette J. Dendritic cells enhance growth and differentiation of CD40-activated B lymphocytes [J]. Journal of Experimental Medicine, 1997, 185(5): 941-952.
[35] Nakayama Y, lnoue Y, Minagawa N. Relationships between S-100 protein-positive cells and clinicopathological factors in patients with colorectal cancer [J]. Anticancer Research, 2003, 23(6):4423-4426.
[36] Morse M A, Lyerly H K. Dendritic cell2 based immunization for cancer therapy [J].Advances in Experimental Medicine and Biology, 2000, 465: 3335-346.
[37]Am be K, Mori M, Enjoji M. S-100 protein-positive dendritic cells in colorectal adenocarcinomas. Distribution and relation to the clinical prognosis [J]. Cancer, 1989, 63(3):496-503.
[38] Tsujitani S, Kakeji Y, Watanabe A, et al. Infiltration of dendritic cells in relation to tumor invasion and dendritic cells in relation to tumor invasion and lymph node metastasis in human gastric cancer [J]. Cancer, 1990, 66(9): 2012-2016.
[39] 王正听,曹雪涛,张明微,等.人肿瘤浸润性树突状细胞的表型分析及原因探讨[J].中国中流生物治疗杂志,1999,6(3):326-329.
[40] Jalili T, Murthy G G. Schiestl R H. Cigatette smoke induces DNA deletions in the mouse embryo [J]. Cancer Research, 1998,58(12): 2633-2638.
[41] Chaux P, Favre N, Martin M, et al. Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats [J]. International Journal of Cancer, 1997, 72(4): 619-624.
[42] Gabrilovich Dl, Ciernik IF, Carbone DP. Dendritic cells in antitumor immune responses 1.Defective antigen presentation in tumor-bearing hosts [J]. Cell Immunology, 1996, 170(1): 101 —110.
[43] Smyth M J, Godfrey D 1, Crapani J A. A fresh look at tumor immunosurveillance and immunotheraphy [J]. Nature Immunology, 2002, 2(4): 293—299.
[44] Lipscomb M F, Masten B J. Dendritic cells: immune regulators in health and disease [J].Physiology Review, 2002, 82(1): 97—130.
[45] Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance [J]. Nature Reviews Cancer, 2005, 5(4): 263—274.
[46] Chaux P, Moutet M. Inflammatory cells infiltrating human colorectal carcinomas express HLA class II but not B7-1 and B7-2 costimulatory molecules of the T cell activation [J].Laboratory Investigation, 1996, 74(5): 975—980.
[47] Troy A, Davidson P, Atkinson C, et al. Phenotypic characterization of the dendritic cell infiltrate in prostate cancer [J]. Journal of Urology, 1998, 160(1): 214—219.
[48] Vermi W, Bonecchi R, Facchetti F, et al. Recruitment of immature plasmacytoid dendritic cells and myeloid dendritic cells in primary cutaneous melanomas [J]. Journal of Pathology, 2003,200 (2): 255-268.
[49] Yanagimoto H, Takai S, Satoi S, et al. Impaired function of circulating dendritic cells in patients with pancreatic cancer [J]. Clinical Immunology, 2005, 114(1): 52-60.
[50] Esche C, Lokshin A, Shurin G V, et al. Tumor's other immune targents: dendritic cells [J].Journal of Leukocyte Biology, 1999, 6(2): 336—344.
[51] Berthier V O, Gaucherand M, Peguet NJ, et al. Human melanoma cells inhibit the earliest differentiation steps of human Langerhans cell precursors but failed to affect the functional maturation of epidermal Langerhans cells [J]. British Journal of Cancer, 2001, 85(12): 1944—1951.
[52] Rudi J, Kuck D, Strand S, et al. Involvement of the CD95 (Apo21/Fas) receptor and ligand system in helicobacter pylori induced gastric epithelial apoptosis [J]. The Journal of Clinical Investigation, 1998, 102(8): 1506-1512.
[53] Niehans G A, Brunner T, Frizelle S P, et al. Human lung carcinomas express Fas ligand [J].Cancer Research, 1997, 57(6): 1007-1012.
[54] Saas P, Walker P R, Hahne M, et al. Fas ligand expression by astrocytoma in vivo:maintaining immune privilege in the brain [J]. Journal of Clinical Investigation, 1997, 99(6):1173-1178.
[55] Bullani R R, Wehrli P, Viard LI, et al. Frequent down regulation of Fas (CD95) expression and function in melanoma [J]. Melanoma Research, 2002, 12(3): 263—270.
[56] Restifo N P. Not so Fas: reevaluating the mechanisms of immuneprivilege and tumor escape [J]. Melanoma Research, 2000, 6(5): 493-495.
[57] Chen S R. Akbar S M, Tanimoto K, et al. Absence of CD83-positive mature and activated dendritic cells at cancer nodules from patients with hepatocellular carcinoma: relevance to hepatocarcinogenesis [J]. Cancer Letter, 2000, 148: 49—57.
[58] Almand B, Resser J R. L indman B, et al. Clinical significance of defective dendritic cell differentiation in cancer [J]. Clinal Cancer Research, 2000, 6: 1755 — 1766.
[59] Troy A J, Davidson P J, Atkinson C H, et al. CD1a dendritic cells predominate in transitional cell carcinoma of bladder and kidney but are minimally activated [J]. Journal of Urology, 1999,161: 1962-1967.
[60] Mailliard R B, Dallal R M. Son Y I, et al. Dendritic cells promote T cell survival or death depending upon their maturation state and presentation of antigen [J]. Immunological Investigations, 2000, 29: 177— 185.
[61] Morandi F, Chiesa S, Bocca P. et al. Tumor mRNA-transfected dendritic cells stimulate the generation of CTL that recognize neuroblastoma-associated antigens and kill tumor cells:immunotherapeutic implications[J].Neoplasia,2006,8(10):833-842.
[62]王斌,萱垣升,李杰芬.灵芝多糖对小鼠脾脏树突状细胞的增殖作用[J].免疫学杂志,2006,22(3):283-285.
[63]王斌,李杰芬,胡岳山.当归多糖对小鼠脾脏树突状细胞增殖的影响[J].解剖学杂志,2005,28(4):403-404.
[64]郭文菁,杨美香,曲迅,等.罗勒多糖对树突状细胞表面分子表达的影响[J].中国免疫学杂志,2006,22(9):827-829.
[65]陈国安,肖希斌,袁利亚,等.当归多糖促进慢性粒细胞白血病细胞性树突状细胞的诱导生成[J].中草药,2004,35(5):531-535.
[66]李罗清,孙圣刚,曹学兵,等.早期脂多糖干预对大鼠树突状细胞表型和功能的影响[J].免疫学杂志,2005,21(3):193-196.
[67]Shao P,L H Zhao,et al.Regulation on maturation and function of dendritic cells by Astragalus mongholicus polysaccharides[J].International Immunopharmacology,2006,6(7):1161-1166.
[68]朱杰,赵鲁杭,陈智.枸杞多糖对小鼠骨髓树突状细胞成熟的影响[J].浙江大学学报:医学版,2006,35(6):648-652.
[69]侯安继,杨占秋,黄菁,等.羧甲基茯苓多糖上调HBV转基因小鼠树突状细胞功能[J].武汉大学学报:理学版,2006,52(6):778-782.
[70]Termeer C,Benedix E Sleeman J,et al.Oligosaccharides of hyaluronan activate dendritic cells via Toll-like receptor 4[J].Journal of Experimental Medicine,2002,195(1):99-111.
[71]Cao L Z,Zhi-Bin Lin Z B.Regulation on maturation and function of dendritic cells by Ganoderma lucidum polysaccharides[J].Immunology Letters,2002,83:163-169.
[72]Park S K,Kim G Y,Lira J Y,et al.Acidic polysaccharides isolated from Phellinus linteusinduce phenotypic and functional maturation of murine dendritic cells[J].Biochemical and Biophysical Research Communication,2003,312(2):449-458.
[73]Kanazawa M,Mori Y,Yoshihara K,et al.Effect of PSK on the maturation of dendritic cells derived from human peripheral blood monocytes[J].Immunology Letters,2004,91(2-3):229-238.
[74]王薇,李萍萍.中药影响树突状细胞抗肿瘤免疫作用的研究进展[J].临床肿瘤学杂志,2007,12(8):624-630.
[75]王俊,周智东,夏大静.香菇多糖增强树突状细胞瘤苗的抗肿瘤作用及其机制研究[J].中国中西医结合杂志,2007,27(1):60-64.
[76]荣微,井欢,刘春英.香菇多糖对荷瘤小鼠树突状细胞功能影响的实验研究[J].中医药学刊,2006,24(4):681-682.
[77]李声方,王兮,桂希恩,等.当归多糖对乙肝病毒转基因小鼠树突状细胞功能状态的影响[J].实用诊断与治疗杂志,2005,19(5):313-314.
[78]黄菁.羧甲基茯苓多糖对HBV转基因小鼠树突状细胞的影响[D].武汉大学,2005.
[79]林秋叶.刺五加水提物抗炎作用及其机制研究[D].大连理工大学,2007.
[80]谢燕霞.芦笋多糖对巨噬细胞的免疫调节研究[D].山东师范大学,2008
[81]Schepetkin I A,Fauikner C L,Nelson-Overton L K,et al.Macrophage immunomodulatory activity of polysaccharides isolated from Juniperus scopolorum[J].International Immunopharmacology,2005,5(13-14):1783-1799.
[82]Schepetkin I A,Quinn M T.Botanical polysaccharides:macrophage immunomodulation and therapeutic potential[J].International Immunopharmacology,2006,6(3):317-333.
[83]Nose M,Terawaki K.Activation of macrophages by crude polysaccharide fraction sobtained from shoots of Glycyrrhiza glabra and hairt roots of Glycyrrhiza uralensis in vitro[J].Biological & Pharmaceutical Bulletin,1998,21(10):1110-1112.
[84]丁航,侯敢,周克元,等.香菇多糖对巨噬细胞一氧化氦和一氧化氮合酶活性的影响[J].广东药学,2003,13(4):32-34.
[85]刘易通,解慧梅,李印君.花粉多糖对血管内皮细胞分泌NO的影响及其免疫增强机制的探讨[J].中国家禽,2006,23(1):50-51.
[86]侯敢,黄迪南,祝其锋.猪荃多糖对小鼠腹腔巨噬细胞一氧化氮生成的影响及其机理[J].中国老年学杂志,2000,20(4):233-235.
[87]侯敢,黄迪南,杨明,等.芦荟多糖对小鼠腹腔巨噬细胞NO生成和iNOS活性的影响[J].湖南中医学院学报,2006,26(3):20-23.
[88]陈伟,林新华,黄丽英,等.库拉索芦荟多糖对小鼠腹腔巨噬细胞一氧化氮生成的影响[J].中国现代应用药学杂志,2006,23(1):17-20.
[89]Alain T,Ziad M.Cytokines in atherosclerosis:pathogenic and regulatory pathways[J].Physiological Reviews,2006,86(2):515-581.
[90]Wu M J,Weng C Y,Wang L,et al.Immunomodulatory mechanism of the aqueous extract of sword brake fern(Pteris ensiformis Burm.)[J].Journal of Ethnopharmacology,2005,98(1-2):73-81.
[91]Luk J M,Lai W,Tam P,et al.Suppression of cytokine production and cell adhesion molecule expression in human monocytic cell line THP-1 by Tripterygium wilfordii polysaccharide moiety [J].Life Science,2000,67:155-63.
[92]Zhang Y,Kiyohara H,Matsumoto T,et al.Fraction-ation and chemical properties of immunomodulating polysaccharides from roots of Dipsacus asperoides[J].Planta,Medica 1997,63:393-399.
[93]Egger S F,Brown G S,Kelsey L S,et al.Hematopoietic augmentation by a β-(1-4)-linked mannan[J].Cancer Immunology,Immunotherapy,1996,43:195-205.
[94]Chen Y J,Shiao M S,Lee S S,et al.Effect of Cordyceps sinensis on the proliferation and differentiation of human leukemic U937 cells[J].Life Science,1997,60:2349-2359.
[95]江振友,林晨,刘小澄,等.灵芝多糖对小鼠体液免疫功能的影响[J].暨南大学学报(医学版),2003,24(2):51-53.
[96]Nair P K,Rodriguez S,Ramachandran R,et al.Immune stimulating properties of a novel polysaccharide from the medicinal plant Tinospora cordifolialnt[J].Immunopharmacol,2004,4(13):1645-1659.
[97]Gangloff S C,Zahringer U,Blondin C,et al.Influence of CD14 on ligand interactions between lipopolysaccharide and its receptor complex[J].Journal of immunology,2005,175(6):3940-3945.
[98]Lacchini A H,Davies A J,Machintosh D,et al.β-1,3-glucan modulates PKC signalling in Lymnaea stagnalis defence cells:a role for PKC in H2O2 production and downstream ERK activation[J].Journal of Experimental Biology,2006,209(24):4829-4840.
[99]孙春艳,赵伯涛,郁志芳,等.芦笋的化学成分及药理作用研究进展[J].中国野生植物资源,2004,23(5):1-5.
[100]Yoon Y D,Kang J S,Han S B,et al.Activation of mitogen-activated protein kinases and AP-1 by polysaccharide isolated from the radix of Platycodon grandiflorum in RAW 264.7 cells [J].International Immunopharmacology,2 2004,4(12):1477-1487.
[101]Zughaier S M,Zimmer S M,Datta A,et al.Differential induction of the toll-like receptor 4-MyD88-dependent and -independent signaling pathways by endotoxins[J].Infect Immunology,2005,73(5):2940-2950.
[102]Nakamura T,Suzuki H,Wada Y,et al.Fucoidan induces nitric oxide production via p38mitogen-activated protein kinase and NF-κB-dependent signaling pathways through macrophage scavenger receptors[J].Biochemical and Biophysical Research Communications,2006,343(1):286-294.
[103]Lim T S,Na K,Choi E M,et al.Immunomodulating activities of polysaccharides isolated from Panax ginseng[J].Journal of Medicinal Food,2004,7(1):1-6.
[104]Gordon S.Pattern recognition receptors:doubling up for the innate immune responses[J].Cell,2002,111(7):927-930.
[105]Jeon Y J,Kim H M.Experimental evidences and signal transduction pathways involved in the activation of NF-kappa B/Rel by angelan in murine macrophages[J].International Immunopharmacology,2001,1(7):1331-1339.
[106]Han S B,Park S H,Lee K H,et al.Polysaccharide isolated from the radix of Platycodon grandiflorum selectively activates B cells and macrophages but not T cells[J].International Immunopharmacology,2001,1(11):1969-1978.
[107]Li S G,Zhang Y Q,Zhao J X.Preparation and suppressive effect of astragalus polysaccharide in glornerulonephritis rats[J].International Immunopharmacology.2007,7(1):23-28.
[108]Lee K Y,Jeon Y J.Macrophage activation by polysaccharide isolated from Astragalus membranaceus[J].International Immunopharmacology,2005.5(7-8):1225-1233.
[109]Jeon Y J,Han S B,Ann K S.Activation of NF-κB/Rel in angelan-stimulated macrophages [J].Immunopharmacology,1999,43(1):1-9.
[110]Escribano J,Az-Guerra M J,Riese H H,et al.In vitro activation of macrophages by a novel proteoglycan isolated from corms of Crocus sativus L[J].Cancer Letter.1999,144(1):107-114.
[111]Hsu M J,Lee S S,Lee S T,et al.Signaling mechanisms of enhanced neutrophil phagocytosis and chemotaxis by the polysaccharide purified from Ganoderma lucidum[J].British Journal of Pharmacology,2003,139(2):289-298.
[112]Zuo J,Jin L,Lv J,et al.Induced NO production and iNOS mRNA expression of macrophage by polysaccharide from Paecilomyces tenuipes[J].Chinese Journal of Biochemical Pharmaceutics,2006,27(4):205-208.
[113]Zhang G L,Wang Y H,Ni W,et al.Hepatoprotective liver injury in mice[J].World Journal of Gastroenterology,2002,8(4):728-733.
[114]Zhang B Q,Hu S J,Qiu L H.Effects of Astragalus membranaceus and its main components on the acute phase endothelial dysfunction induced by homocysteine[J].Vascular Pharmacology,2007,46(4):278-285.
[115]候宽昭.中国种子植物科属辞典[M].北京:科学出版社,1982.
[116]郑太坤.中国车前草的形态鉴定[J].中国中药杂志,1990,7:6.
[117]郑太坤.中药车前的研究概况[J].辽宁中医杂志,1985,(8):41.
[118]中华人民共和国卫生部药典委员会.中华人民共和国药典(一部)[M].北京:人民卫生出版社,1990.
[119]王东.车前子多糖的研究[D].辽宁中医学院,2003.
[120]Masashi T,Maemi Y,Kazuyo 1.Plant mucilages.ⅩⅪⅩ.Isolation and characterization of a mucous polysaccharide,"Piantago-mucilage A," from the seeds of Plantago major var.asiatica [J].Chemical & Pharmaceutical Bulletin,1981,29(10):2877-2884.
[121]SamuelsenA B,Lund I,Djahromi J M,et al.Structural features and anti-complementary activity of some heteroxylan polysaccharide fractions from the seeds of Plantago major L[J].Carbohydrate Polymers,1999,38:133-143.
[122]Miyase T,Ishino M,Akahori C,et al.Phenylethanoid Glycosides from Plantago asiatica[J].Phytochemistry,1991,30(6):2015-2018.
[123]Nishibe S,Kodama A,Noguchi Y,et al.Phenolic compounds from seeds of Plantago ovata and P.psyllium[J].Natural Medicines(Tokyo,Japan),2001,55(5):258-261.
[124]Li L,Tsao R,Liu Z Y,et al.Isolation and purification of acteoside and isoacteoside from Plantago psyllium L.by high-speed counter-current chromatography[J].Journal of Chromatography A,2005,1063:161-169.
[125]高明哲,张惠,林立,等.车前子苷的提取分离[J].辽宁中医学院学报,2003,5(2):157.
[126]Patel R B,Rana N G,Patel M R,et al.Chromatographic screening of proteins of seeds of Plantago ovata Forsk[J].Indian Drugs Pharmacology.,1981,16(1):3-5.
[127]付志红.车前子营养与活性成分及其保健功能研究[D].南昌大学,2005.
[128]Rilka T,Ljubka E,Nedjalka H,et al.Iridoid patterns of genus Plantago L.and their systematic significance[J].Journal of Biosciences,2002,57:42-50.
[129]Marteau P,Flourie B,Cherbut C,et al.Digestibility and bulking effect of ispaghula husks in healthy humans[J].Gut,1994,35(12):1747-1752.
[130]Marlett J A,Kajs T M,Fischer M H.An unfermented gel component of psyllium seed husk promotes laxation as a lubricant in humans[J].The American Journal Of Clinical Nutrition,2000,72(3):784-789.
[131]Marlett J A,Fischer M H.A poorly fermented gel from psyllium seed husk increases excreta moisture and bile acid excretion in rats[J].Journal of Nutrition,2002,132(9):2638-2643.
[132]Marlett J A,Fischer M H.The active fraction of psyllium seed husk[J].The Proceedings of The Nutrition Society,2003,62(1):207-209
[133]Edwards C A,Bowen J,Brydon W G,et al.The effects of ispaghula on rat caecal fermentation and stool output[J].British Journal of Nutrition,1992,68(2):473-482
[134]张今强.车前子多糖微丸缓泻制剂的研究[D].辽宁中医学院,2005.
[135]吴光杰,田颖刚,谢明勇,等.车前子多糖对便秘模型小鼠通便作用的研究[J].食品科学,2007,28(10):514-516.
[136]Karen W,Peter B.Reduction of glycenic and lipid levels in db/db diabetic mice by Psyilium plant fiber[J].Diabetes,1989,38(12):1528-1533.
[137]Frati Munari A C,Benitez Pinto W,Raul Ariza Andraca C,et al.Lowering glycemic index of food by acarbose and Plantago psyllium mucilage[J].Archives Of Medical Research,1998,29(2):137-141.
[138]Narayan R.Meet Psyllium:A fiber product with potential cardioprotective Effects[J].Nutrition Noteworthy,2005,7,Article 3.
[139]栗艳彬.车前子胶调血脂及降血糖作用的实验研究[D].辽宁中医学院,2004.
[140]Tomoda M,Takada K,Shimizu N,et al.Reticuloendothelial system-potentiating and alkaline phosphatase-inducing activities of Plantago-mucilage A,the main mucilaget from the seed of Plantago asiatica,and its five modification products[J].Chemical & Pharmaceutical Bulletin,1991,39(8):2068-2071.
[141]谢小梅,付志红,谢明勇,等.精制车前子多糖对小鼠免疫功能的影响[J].第三届全国中医药免疫学术研讨会论文汇编.中国,湖南.2006;18-22.
[142]张振秋,李锋,孙兆妹,等.车前子的药效学研究[J].中药材,1996,19(2):87-89.
[143]Deters A M,Schroder K R,Smiatek T,et.al.Ispaghula(Plantago ovata) seed husk polysaccharides promote proliferation of human epithelial cells(skin keratinocytes and fibroblasts)via enhanced growth factor receptors and energy production[J].Planta Medical,2005,71:33-39.
[144]Westerhof W,Das P K,Middelkoop E,et al.Mucopolysaccharides from psyllium involved in wound heating(Abstract)[J].Drugs under Experimental and Clinic Research,2001,27(5-6):165-175.
[145]韩强,林惠芬,朱玲莉.几种中药提取物对酪氨酸酶活性的抑制[J].香料香精化妆品,1998,4:22-23.
[146]Shortman K,Caux c.dendritic cells development:multiple pathways to nature's adjuvants [J].Stem Cells.1997,15(6):409-419.
[147]Korting H C,Herzinger T.Hartinger A,et al.Discrimination of the irritancy potential of surfactants in vitro by two cytotoxicity assays using normal human keratinocytes,HaCaT cells and 3T3 mouse fibroblasts:correlation with in vivo data from a soap chamber assay[J].Journal Dermatological Science,1994,119-129.
[148]张卓然.培养细胞学与细胞培养技术[M].上海:上海科学技术出版社,2004.
[149]董伟华,郑智敏,刘桂亭.中药大黄、槐米、红花提取物的致突变作用[J].河南医科大学学报,1991,2(64):330-333.
[150]张建清,苏城玉,权玉玲,等.四种补益中药的急性、毒性利致突变性研究[J].现代预防医学,1999,2(611:26-28.
[151]谢建华.青钱柳多糖的分离纯化与结构解析及其生物活性研究[D].南昌大学,2007.
[152]Nie S P,Xie M Y,Fu Z H,et al.Study on the purification and chemical compositions of tea glycoprotein[J].Carbohydrate Polymers,2008,71:626-633.
[153]殷军艺,聂少平,付志红,等.大粒车前子多糖分离、纯化及单糖组成分析[J].食品科学,2008,29(09):529-532.
[154]周超.车前子多糖分离、纯化及功能特性研究[D].南昌大学,2007.
[155]万茵.车前子多糖、黄酮和苯乙醇苷类的纯化、结构解析及其活性功能研究[D].南昌大学,2007.
[156]戴建国,李超,黄培林.高密度壳聚糖对L-929细胞的毒性研究[J].东南大学学报(医学版),2008,27(1):54-57.
[157]翟中和,王喜忠,丁明孝.细胞生物学[M].北京:高等教育出版社,2000.
[158]Inaba k,Inaba M,Romani N,et al.Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulation factor[J].Journal of Experimental Medicine,1992,176:1693-1702.
[159]夏俊波,吴奎,孙鲲,等.小鼠骨髓来源的树突状细胞体外扩增与鉴定[J].第三军医大学学报,2005,27(10):942-944.
[160]Basak S K,Harui A,Stolina M,et al.Increased dendritic cell number and function folowing continuous in vivio infusion of granulocyte macrophage-colony-stimulation factor and interleukin-4[J].Blood,2002,99(8):2869-2879.
[161]罗建飞,陈必成,陈忠华.小鼠骨髓来源树突状细胞的分离与扩增培养[J].微循环学杂志,2005,15(1):29-31.
[162]Markowicz S,Engleman E G.Granulocyte-macrophage colony-stimulating factor promotes differentiation and survival of human perpheral blood dendritic cells in vivo[J].Journal of Clinical Investigation,1990,85:955-961.
[163]Gang M Z,Ying K.Cytokines in the generation and maturation of dendritic cells:recent advances[J].European Cytokine Network,2002;13(2):186-199.
[164]Aerts-Toegaert C,Heirman C,Tuyaerts S.CD83 expression on dendritic cells and T cells:correlation with effective immune responses[J].European Journal of Immunology,2007,37:686-695.
[165]Lutz M B,Suri R M,Niimi M,et al.Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo[J].European Journal of Immunology,2000,30(7):1813-1822.
[166]Cella M,Sallusto F,Lanzavecchia A,et al.Origin,maturation and antigen presenting function of dendritic cells[J].Current opinion in Immunology,1997,9(1):10-16.
[167]黄贝贝,肖凤仪,张文平,等.青钱柳对小鼠免疫功能的影响[J].江西中医学院学报,2004,16(5):59-60.
[168]汪东风,李俊,王常红,等.茶叶多糖的组成及免疫活性研究[J].茶叶科学,2000,20(1):45-50.
[169]R M Steinman.The dendritic cell system and its role in immunogenicity[J].Annual Review of Immunology,1991,9:271-296.
[170]Lin Y L,Liang Y,Lee S S,et al.Polysaccharide purified from Ganoderma lucidum induced activation and maturation of human monocyte-derived dendritic cells by the NF-κB and p38mitogen-activated protein kinase-pathways[J].Journal of Leukocyte Biology Volume,2005,78:533-543.
[171]Roake J A,Rao A S,Morris P J,et al.Dendritic cell loss from nonlymphoid tissues after systemic administration of lipopolysaccharide,tumor necrosis factor,and interleukin 1[J].Journal of Experimental Medicine,1995,181:2237-2247.
[172]Sallusto F,Celia M,Danieli C,et al.Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class Ⅱcompartment:downregulation by cytokines and bacterial products[J].Journal of Experimental Medicine,1995,182:389-400.
[173]O'Sullivan B J,Thomas R.CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-κB[J].Journal of Immunology,2002,168:5491-5498.
[174]Xie J,Qian J,Wang S,et al.Novel and detrimental effects of lipopolysaccharide on in vitro generation of immature dendritic cells:involvement of mitogen-activated protein kinase p38[J].Journal of Immunology,2003,171:4792-4800.
[175]Omarsdottir S,Olafsdottir E S,Freysdottir J,et al.Immunomodulating effects of lichen-derived polysaccharides on monocyte-derived dendritic cells[J].International Immunopharmacology,2006,6:1642-1650.
[176]Kim J Y,Yoon Y D,Ahn J M.Angelan isolated from Angelica gigas Nakai induces dendritic cell maturation through toll-like receptor 4[J].International Immunopharmacology,2007,7:78-87.
[177]Kim G Y,H Cho,Ahn S C,et al.Resveratrol inhibits phenotypic and functional maturation of murine bone marrow-derived dendritic cells[J].International Immunopharmacology,2004,4:245-253.
[178]Liu Q.Chen T,Chen G,et al.Immunosuppressant triptolide inhibits dendritic cell-mediated chemoattraction of neutrophils and T cells through inhibiting Stat3 phospborylation and NF-κB activation[J].Biochemical and Biophysical Research Communications,2006,345:1122-1130.
[179]Zhou L J,Tedder T F.Human blood dendritic cells selectively express CD83,a member of the immunoglobulin superfamily[J].Journal of Immunology,1995,154(8):3821-3835.
[180]Adams S,O'Neill D W,Bhardwaj N.Recent advances in dendritic cell biology[J].Journal of Clinical Immunology,2005,25(3):177-188.
[181] Wu L, Vremec D, Ardavin C, et al. Mouse thymus dendritic cells: kinetics of development and changes in surface markers during maturation [J]. European Journal of Immunology, 1995,25(2): 418-425.
[182] Pieters J. MHC class 11 restricted antigen presentation [J]. Current Opinion in Immunology,1997, 9(1):89-96.
[183] Lu L, Qian S, Hershberger P A, et al. Fas ligand (CD95L) and B7 expression on dendritic cells provide counter-regulatory signals for T cell survival and proliferation [J]. Journal of Immunology, 1997, 158(12): 56-76.
[184] Koch R. High level 1L-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10 [J]. Journal of Experimental Medicine, 1996, 184: 741-747.
[185] Moore K W, de Waal Malefyt R, Coffman R L, et al. Interleukin-10 and interleukin-10 receptor [J].Annual Review Immunology, 2001, 19 : 683—765.
[186] Bogdan C. Nitric oxide and the immune response [J]. Nature Immunology, 2001, 2: 907—916.
[187] Philip S. IL-12: initiation cytokine for cell-mediated immunity [J]. Science, 1993, 260:496-497.
[188] Macatonia S E, Hosken N A, Litton M, et al. Dendritic cells produce IL-12 and direct the decelopment of Thl cells from naive CD4+ T cells [J]. Journal of Immunology, 1995, 154:5071-5079.
[189] Perona-Wright G, Jenkins S J, Crawford A, et al. Distinct sources and targets of IL-10 during dendritic cell-driven Thl and Th2 responses in vivo[J]. European Journal of Immunology,2006,36(9):2367-2375.
[190] Ebner S, Ratzinger G, Krosbacher B, et al. Production of IL-12 by human monocyte-derived dendritic cells is optimal when the stimulus is given at the onset of maturation, and is further enhanced by IL-4 [J]. The Journal of Immunology, 2001, 166: 633—641.
[191] Lamont A G, Adorini L. IL-12, a key cytokine in immune regulation [J]. Immunol Today, 1996, 17(5): 214-217.
[192] Austyn J M, Kupiec-Weglinski J W, Hankins D F, et al. Migration patterns of dendritic cells in the mouse. Homing to T cell-dependent areas of spleen, and binding within marginal zone [J].Journal of Experimental Medicine, 1998, 67: 646—651.
Austyn J M. Migration patterns of dendritic leukocytes [J]. Res. Immunol, 1 989, 140: 898—902.
[193] Shortman K, Liu Y J. Mouse and human dendritic cell subtypes [J]. Nature Reviews Immunology. 2002, 2(3) :151 —161.
[194] Wilson N S, Villadangos J A. Regulation of antigen presentation and cross-presentation in the dendritic cell network:facts.hypothesis,and immunological implications [J]. Advances in immunology, 2005, 86: 241-305.
[195] Guermonprez P, Valladeau J, Zitvogel L, et al. Antigen presentation and T cell stimulation by dendritic cells [J]. Annual Review of Immunology, 2002, 20: 621 —667.
[196]Thery C,Amigorena S.The cell biology of antigen presentation in dendritic cells[J].Current Opinion in Immunology,2001,13(1):45-51.
[197]Jonathan M,Austyn,Ralph M,et al.Dendritic cells initiate a two-stage mechanism for T iymphyocyte proliferation[J].The Journal of Experimental Medicine,1983,157(4):1101-1115.
[198]Dieu-Nosjean M C,Vicari A,Lebecque S,et al.Reuglation of dendritic cell trafficking:a proeess that involves the participation of selective chemokines[J].Journal of Leukocyte Biology,1999,66:252-262.
[199]Setinman R M.The dendritic cell system and its role in immunogencity[J]Annual Review of Immunology,1991,9:271-296.
[200]Sozzani S,Allvaena,P Vecchi A,et al.Chemokines and dendritic cell traffic[J].Journal of Clinical Immunology,2000,20:151-160.
[201]Sallusto F,Palermo B,Lenig D,et al.Distinct Patterns and kinetics of chemokine production regulate dendritic cell function[J].European Journal of Immunology,1999,29(5):1617-1625.
[202]Caux C,Ait-Yahia S,Chemin K,et al.Dendritic cell biology and regulation of dendritic cell trafficking by chemokines[J].Springer Semin Immunopathol,2000,22(4):345-369.
[203]Forster R,Schubel A,Beritefld D,et.al.CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoidorgans[J].Cell,1999,99:23-33.
[204]张卓然.培养细胞学与细胞培养技术[M].上海:上海科学技术出版社,2004.
[205]Dieu M C,Vanbervlietn B,Vieari A,et al.Seleetive recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anato-mic sites[J].The Journal of Experimental Medicine.1998,188(2):373-386.
[206]Cyster J G.Chemokines and the homing of dendritic cells to theTcell areas of lymphoid organ[J].The Journal of Experimental Medicine,1999,189(3):447-450.
[207]Hirao M,Onai N,Hiroishi K,et al.CC chemokine receptor-7 on dendritic cells is induced after interaetion with apoptotic tumor cells:critical role in migration from the tumor site to draining lymph nodes[J].Cancer Research,2000,60(8):2209-2217.
[208]Sato K,Kawasaki H,Nagayama H,et al.Signaling events following chemokine receptor ligation in human dendrtic cells atdiffrent developmenta lstages[J].International Immunology,2001,13(2):167-179.
[209]长琦,许有玲,等.抗肿瘤植物药及其有效成分[M].北京:中国中医药出版社,1997.
[210]Ishida T,Oyma T,Cabone D P,et al.Defective function of Langerhans cells in tumor-bearing animals is the result of defective maturation from hemopoietic progenitors [J].Journal of Immunology,1998,161(9):4842-4851.
[211]Dmitry I,Frank C,David P,et al.Dendritic cells in antitumor immune responses defective antigen presentation in tumor-bearing hosts[J].Cell Immunology,1996,170(1):101-103.
[212]何彦丽,苏俊芳.中药多糖抗肿瘤免疫研究的新视角——对树突状细胞的影响[J].中药药理与临床,2002,18(4):47-49.
[213]Matsumoto M,Fu Y X,Molina H,et al.Distinct roles of lymphotoxin and the typel tumor necrosis factor(TNF) reeptor in the catablishment of folliuclar dendrtic cells from non-bone marrow dendritic cells[J].The Journal of Experimental Medicine,1997,186:1997-2004.
[214]Chiang L C,Chiang W,Chang M Y,et al.In vitro cytotoxic,antiviral and immunomodulatory effects of Plantago major and Plantago asiatica[J].American Journal of Chinese Medicine,2003,31(2):225-234.
[215]Figdor C G,de Vriesl J,Lesterhuis W J,et al.Dendritic cell Immuno-therapy:mapping the way[J].Nature Medicine,2004,10(5):475-480.
[216]Jeofford M,Marskovsky E,Cebon J,et al.The use of dendritic cells in cancer therapy[J].The Lancet Oncoiogy,2001,2(6):343-353.
[217]Nair S K,Snyder D,Rouse B T,et al.Regression of tumors in mice vaccinated with professional antigen-presenting cells pulsed with tumor extracts[J].International Journal of Cancer,1997,70:706-715.
[218]Hattori TS,Komatsu N,Shichijo S,et al.Protein-bound polysaccharide K induced apoptosis of the human Burkitt lymphoma cell line,Namalwa[J].Biomedicine &Pharmacotherapy 58(2004):226-230.
[219]De V S,Arredouani M,Ade M,et al.Uptake and presentation of malignant glioma tumor cell lysates by monocyte-derived dendritic cells[J].Cancer Immunology lmmunotherapy,2005,54(4):372-382.
[220]Kumamoto T,Morita A,Takashima A.Recent advances in dendritic cell vaccines for cancer treatment[J].the Journal of Dermatology,2001,28(11):658-662.
[221]Samuelsen A B.The traditional uses,chemical constituents and biological antivities of Plantago major L.[J].Journal of Ethnopharmacology,2000,71:1-21.
[222]Ji Z,Tang Q,Zhang J,et al.Immunomodulation of RAW264.7 macrophages by GLIS,a proteopolysaccha-ride from Ganoderma lucidum[J].Journal of Ethnopharmacology,2007,112(3):445-450.
[223]游育红,林志彬.灵芝多糖肽对小鼠腹腔巨噬细胞一氧化氮产生的影响[J].中国药理学通报,2004,20(12):1398-1401.
[224]李俊,黄艳,廖日权,等.罗汉果多糖对小鼠免疫功能的影响[J].中国药理学通报,2008,24(9):1237-1240.
[225]Shishodia S,Aggarwal B B.Nuclear factor-kappaB Activation:A question of life or death [J].The Journal of Steroid Biochemistry and Molecular Biology,2002,35(1):28-34.
[226]Zhang L,Tizard I R.Activation of a mouse macrophage cell line by acemannan:the major carbohydrate fraction from Aloe vera gel[J].Immunopharmacology,1996,35(2):119-128.
[227]Karaca K,Sharma J M,Nordgren R.Nitric oxide production by chicken macrophages activated by acemannan,a complex carbohydrate extracted from Aloe vera[J].International Journal of Immunopharmacology,1995,17(3):183-188.
[2]Dannull J,Cerny T,Ackermann DK,et al.Current status of dendritic cell-based tumor vaccination[J].Onkologie,2000,23(6):544-551.
[3]Hows J M,Marsh J C,Bradley B A,et al.Human cord blood:a source of transplantable stem cells[J].Bone Marrow Transplant,1992,9(Suppl 1):105-108.
[4]Oki M,Ando K,Hagihara M,et al.Efficient lentiviral transduction of human cord blood CD34~+ cells followed by their expansion and differentiation into dendritic cells[J].Experimental Hematology,2001,29(10):1210-1217.
[5]Klinkert W E,La Baddie J H,Bowers E.Accessory and stimulating properties of dendritic cells and macrophages isolated from various rat tissues[J].Journal of Experimental Medicine,1982,156(1):1-19.
[6]许亚明,迟诏丞,任明,等.树突状细胞的研究进展[J].吉林医学,2008,29(13):1127-1130.
[7]Rao M S,Mattson M P.Stem cells and aging:expanding the possibilities[J].Mechanisms of Ageing and Development,2001,122(7):713-734.
[8]侯治富,郭楠,高申,等.人外周血树突状细胞的诱导与鉴定[J].吉林大学学报(医学版),2005,31(5):657-659.
[9]Pea-Cruz V,Ito S,Oukka M,et al.Extraction of human Langerhans cells:a method for isolation of epidermis-resident dendritic cells[J].Journal of Immunological Methods,2001,255(1-2):83-91.
[10]郑茂荣,范青源,谢勇,等.Percoll密度梯度离心技术分离纯化豚鼠表皮Langerhans细胞[J].中华皮肤科杂志,1995,28(5):296-298.
[11]舒赛男,魏来,方峰,等.免疫磁珠分选系统在分离大鼠骨髓干细胞群中的应用[J].标记免疫分析与临床,2006,13(1):35-37.
[12]Alessandro A,Stephanie H,Michael F,et al.F.100.GM-CSF induced maturation of dendritic cells is associated with GSK-3beta Inhibition[J].Clinical Immunology,2008,127(1):76.
[13]Markowicz S,Engleman E G.Granulocyte-macrophage colony-stimulating factor promotes differentiation and survival of human perpheral blood dendritic cells in vivo[J].Journal of Clinical Investigation[J],1990,85:955-61
[14]熊菲.影响树突状细胞分化成熟因素的研究进展[J].国外医学免疫学分册,2005,28(2)107-111.
[15]Santini S M,Di Pucchio T,Lapenta C.et al.A new type Ⅰ IFN-mediated pathway for the rapid differentiation of monocytes into highly active dendritic cells[J].Stem Cells,2003,21(3):357-362.
[16] Reid C D. The dendritic cell lineage in haemopoiesis [J]. British Journal of Haematology,1997, 96(2): 217-223.
[17] Lutz M B, Schnare M, Menges M, et al. Differential functions of IL-4 receptor types I and II for dendritic cell maturation and IL-12 production and their dependency on GM-CSF [J]. Journal of Immunology, 2002, 169(7): 3574-3580.
[18] Jacobsen S E, Ruscetti F W, Dubois C M, et al. Tumor necrosis factor a directly and indirectly regulates hematopoietic progenitor cell proliferation: role of colony-stimulating factor receptor modulation [J]. Journal of Experimental Medicine, 1992, 175(6): 1795—1772.
[19] Chen X, Regn S, Raffegerst S, et al. Interferon alpha in combination with GM-CSF induces the differentiation of leukaemic antigen-presenting cells that have the capacity to stimulate a specific anti-leukaemic cytotoxic T-cell response from patients with chronic myeloid leukemia [J].British Journal of Haematology, 2000, 111(2): 596—607.
[20] Urn H D, Cho Y H, Kimdo K, et al. TNF-alpha suppresses dendritic cell death and the production of reactive oxygen intermediates induced by plasma withdrawal [J]. Experimental Dermatology, 2004, 13(5): 282-288.
[21]裴雪涛,王立生,徐黎,等.CD34+造血祖细胞的定向诱导分化研究[J].中华血液学杂志,1998,19(6):289-293．
[22] Piacibello W, Sanavio F, Garetto L, et al. Differential growth factor requirement of primitives cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation [J]. Leukemia, 1998, 12(5): 718—721.
[23] Maraskovsky E, Daro E, Roux E, et al. In vivo generation of human dendritic cell subsets by Flt3ligand [J]. Blood, 2000, 96(3): 878-884.
[24] Riedl E, Strobl H, Majdic O, et al. TGF-beta1 promotes in vitro generation of dendritic cells by protecting progenitor cells from apoptosis [J]. Journal of Immunology, 1997, 158(4): 1591 —1597.
[25] Allavena P, Piemonti L, Longoni D, et al. IL-10 prevents the differentiation of monocytes to dendritic cells but promotes their maturation to macrophages [J]. European Journal of Immunology, 1998, 28(1): 359-369.
[26] Moore K W, de Waal Malefyt R, Coffman RL, et al. Interleukin-10 and the interleukin-10 receptor [J]. Annual Review of Immunology. 2001. 19: 683—765.
[27] Shortman K, Caux C. dendritic cells development:multiple pathways to nature's adjuvants [J].Stem Cells, 1997, 15(6): 409-419.
[28] Thomas R, Davis L, Lipsley P E. Comparative accessory cell of human peripheral blood DC and monocytes [J]. Journal of Immunology. 1993, 151(12): 6840-6852.
[29] Banchereau J, Steinman R M. Dendritic cells and the control of immunity [J]. Nature, 1998,392:245-252.
[30] Hart D N J. Dendritic cells: unique leukocyte populations which control the primary immune response [J]. Blood, 1997, 90(9): 3245-3287.
[31] Brocker T, Riedinger M. Karjalainen K. Targeted expression of majorhisto compatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo [J]. Journal of Experimental Medicine, 1997, 185(3):541-550.
[32] Brocker T. Survival of mature CD4 T lymphocytes is dependent on major histocompatibility comphex class II-expressing dendritic cells [J]. Journal of Experimental Medicine, 1997, 186:1223-1232.
[33] Fayette J, Dubois B, Vandenabeele S. et al. Human dendritic cells skew isotype switching of CD40 activated naive B cells towards IgA 1 and IgA2 [J]. Journal of Experimental Medicine,1997,185:1909-1918.
[34] Dubois B, Vanbervliet B, Fayette J. Dendritic cells enhance growth and differentiation of CD40-activated B lymphocytes [J]. Journal of Experimental Medicine, 1997, 185(5): 941-952.
[35] Nakayama Y, lnoue Y, Minagawa N. Relationships between S-100 protein-positive cells and clinicopathological factors in patients with colorectal cancer [J]. Anticancer Research, 2003, 23(6):4423-4426.
[36] Morse M A, Lyerly H K. Dendritic cell2 based immunization for cancer therapy [J].Advances in Experimental Medicine and Biology, 2000, 465: 3335-346.
[37]Am be K, Mori M, Enjoji M. S-100 protein-positive dendritic cells in colorectal adenocarcinomas. Distribution and relation to the clinical prognosis [J]. Cancer, 1989, 63(3):496-503.
[38] Tsujitani S, Kakeji Y, Watanabe A, et al. Infiltration of dendritic cells in relation to tumor invasion and dendritic cells in relation to tumor invasion and lymph node metastasis in human gastric cancer [J]. Cancer, 1990, 66(9): 2012-2016.
[39] 王正听,曹雪涛,张明微,等.人肿瘤浸润性树突状细胞的表型分析及原因探讨[J].中国中流生物治疗杂志,1999,6(3):326-329.
[40] Jalili T, Murthy G G. Schiestl R H. Cigatette smoke induces DNA deletions in the mouse embryo [J]. Cancer Research, 1998,58(12): 2633-2638.
[41] Chaux P, Favre N, Martin M, et al. Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats [J]. International Journal of Cancer, 1997, 72(4): 619-624.
[42] Gabrilovich Dl, Ciernik IF, Carbone DP. Dendritic cells in antitumor immune responses 1.Defective antigen presentation in tumor-bearing hosts [J]. Cell Immunology, 1996, 170(1): 101 —110.
[43] Smyth M J, Godfrey D 1, Crapani J A. A fresh look at tumor immunosurveillance and immunotheraphy [J]. Nature Immunology, 2002, 2(4): 293—299.
[44] Lipscomb M F, Masten B J. Dendritic cells: immune regulators in health and disease [J].Physiology Review, 2002, 82(1): 97—130.
[45] Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance [J]. Nature Reviews Cancer, 2005, 5(4): 263—274.
[46] Chaux P, Moutet M. Inflammatory cells infiltrating human colorectal carcinomas express HLA class II but not B7-1 and B7-2 costimulatory molecules of the T cell activation [J].Laboratory Investigation, 1996, 74(5): 975—980.
[47] Troy A, Davidson P, Atkinson C, et al. Phenotypic characterization of the dendritic cell infiltrate in prostate cancer [J]. Journal of Urology, 1998, 160(1): 214—219.
[48] Vermi W, Bonecchi R, Facchetti F, et al. Recruitment of immature plasmacytoid dendritic cells and myeloid dendritic cells in primary cutaneous melanomas [J]. Journal of Pathology, 2003,200 (2): 255-268.
[49] Yanagimoto H, Takai S, Satoi S, et al. Impaired function of circulating dendritic cells in patients with pancreatic cancer [J]. Clinical Immunology, 2005, 114(1): 52-60.
[50] Esche C, Lokshin A, Shurin G V, et al. Tumor's other immune targents: dendritic cells [J].Journal of Leukocyte Biology, 1999, 6(2): 336—344.
[51] Berthier V O, Gaucherand M, Peguet NJ, et al. Human melanoma cells inhibit the earliest differentiation steps of human Langerhans cell precursors but failed to affect the functional maturation of epidermal Langerhans cells [J]. British Journal of Cancer, 2001, 85(12): 1944—1951.
[52] Rudi J, Kuck D, Strand S, et al. Involvement of the CD95 (Apo21/Fas) receptor and ligand system in helicobacter pylori induced gastric epithelial apoptosis [J]. The Journal of Clinical Investigation, 1998, 102(8): 1506-1512.
[53] Niehans G A, Brunner T, Frizelle S P, et al. Human lung carcinomas express Fas ligand [J].Cancer Research, 1997, 57(6): 1007-1012.
[54] Saas P, Walker P R, Hahne M, et al. Fas ligand expression by astrocytoma in vivo:maintaining immune privilege in the brain [J]. Journal of Clinical Investigation, 1997, 99(6):1173-1178.
[55] Bullani R R, Wehrli P, Viard LI, et al. Frequent down regulation of Fas (CD95) expression and function in melanoma [J]. Melanoma Research, 2002, 12(3): 263—270.
[56] Restifo N P. Not so Fas: reevaluating the mechanisms of immuneprivilege and tumor escape [J]. Melanoma Research, 2000, 6(5): 493-495.
[57] Chen S R. Akbar S M, Tanimoto K, et al. Absence of CD83-positive mature and activated dendritic cells at cancer nodules from patients with hepatocellular carcinoma: relevance to hepatocarcinogenesis [J]. Cancer Letter, 2000, 148: 49—57.
[58] Almand B, Resser J R. L indman B, et al. Clinical significance of defective dendritic cell differentiation in cancer [J]. Clinal Cancer Research, 2000, 6: 1755 — 1766.
[59] Troy A J, Davidson P J, Atkinson C H, et al. CD1a dendritic cells predominate in transitional cell carcinoma of bladder and kidney but are minimally activated [J]. Journal of Urology, 1999,161: 1962-1967.
[60] Mailliard R B, Dallal R M. Son Y I, et al. Dendritic cells promote T cell survival or death depending upon their maturation state and presentation of antigen [J]. Immunological Investigations, 2000, 29: 177— 185.
[61] Morandi F, Chiesa S, Bocca P. et al. Tumor mRNA-transfected dendritic cells stimulate the generation of CTL that recognize neuroblastoma-associated antigens and kill tumor cells:immunotherapeutic implications[J].Neoplasia,2006,8(10):833-842.
[62]王斌,萱垣升,李杰芬.灵芝多糖对小鼠脾脏树突状细胞的增殖作用[J].免疫学杂志,2006,22(3):283-285.
[63]王斌,李杰芬,胡岳山.当归多糖对小鼠脾脏树突状细胞增殖的影响[J].解剖学杂志,2005,28(4):403-404.
[64]郭文菁,杨美香,曲迅,等.罗勒多糖对树突状细胞表面分子表达的影响[J].中国免疫学杂志,2006,22(9):827-829.
[65]陈国安,肖希斌,袁利亚,等.当归多糖促进慢性粒细胞白血病细胞性树突状细胞的诱导生成[J].中草药,2004,35(5):531-535.
[66]李罗清,孙圣刚,曹学兵,等.早期脂多糖干预对大鼠树突状细胞表型和功能的影响[J].免疫学杂志,2005,21(3):193-196.
[67]Shao P,L H Zhao,et al.Regulation on maturation and function of dendritic cells by Astragalus mongholicus polysaccharides[J].International Immunopharmacology,2006,6(7):1161-1166.
[68]朱杰,赵鲁杭,陈智.枸杞多糖对小鼠骨髓树突状细胞成熟的影响[J].浙江大学学报:医学版,2006,35(6):648-652.
[69]侯安继,杨占秋,黄菁,等.羧甲基茯苓多糖上调HBV转基因小鼠树突状细胞功能[J].武汉大学学报:理学版,2006,52(6):778-782.
[70]Termeer C,Benedix E Sleeman J,et al.Oligosaccharides of hyaluronan activate dendritic cells via Toll-like receptor 4[J].Journal of Experimental Medicine,2002,195(1):99-111.
[71]Cao L Z,Zhi-Bin Lin Z B.Regulation on maturation and function of dendritic cells by Ganoderma lucidum polysaccharides[J].Immunology Letters,2002,83:163-169.
[72]Park S K,Kim G Y,Lira J Y,et al.Acidic polysaccharides isolated from Phellinus linteusinduce phenotypic and functional maturation of murine dendritic cells[J].Biochemical and Biophysical Research Communication,2003,312(2):449-458.
[73]Kanazawa M,Mori Y,Yoshihara K,et al.Effect of PSK on the maturation of dendritic cells derived from human peripheral blood monocytes[J].Immunology Letters,2004,91(2-3):229-238.
[74]王薇,李萍萍.中药影响树突状细胞抗肿瘤免疫作用的研究进展[J].临床肿瘤学杂志,2007,12(8):624-630.
[75]王俊,周智东,夏大静.香菇多糖增强树突状细胞瘤苗的抗肿瘤作用及其机制研究[J].中国中西医结合杂志,2007,27(1):60-64.
[76]荣微,井欢,刘春英.香菇多糖对荷瘤小鼠树突状细胞功能影响的实验研究[J].中医药学刊,2006,24(4):681-682.
[77]李声方,王兮,桂希恩,等.当归多糖对乙肝病毒转基因小鼠树突状细胞功能状态的影响[J].实用诊断与治疗杂志,2005,19(5):313-314.
[78]黄菁.羧甲基茯苓多糖对HBV转基因小鼠树突状细胞的影响[D].武汉大学,2005.
[79]林秋叶.刺五加水提物抗炎作用及其机制研究[D].大连理工大学,2007.
[80]谢燕霞.芦笋多糖对巨噬细胞的免疫调节研究[D].山东师范大学,2008
[81]Schepetkin I A,Fauikner C L,Nelson-Overton L K,et al.Macrophage immunomodulatory activity of polysaccharides isolated from Juniperus scopolorum[J].International Immunopharmacology,2005,5(13-14):1783-1799.
[82]Schepetkin I A,Quinn M T.Botanical polysaccharides:macrophage immunomodulation and therapeutic potential[J].International Immunopharmacology,2006,6(3):317-333.
[83]Nose M,Terawaki K.Activation of macrophages by crude polysaccharide fraction sobtained from shoots of Glycyrrhiza glabra and hairt roots of Glycyrrhiza uralensis in vitro[J].Biological & Pharmaceutical Bulletin,1998,21(10):1110-1112.
[84]丁航,侯敢,周克元,等.香菇多糖对巨噬细胞一氧化氦和一氧化氮合酶活性的影响[J].广东药学,2003,13(4):32-34.
[85]刘易通,解慧梅,李印君.花粉多糖对血管内皮细胞分泌NO的影响及其免疫增强机制的探讨[J].中国家禽,2006,23(1):50-51.
[86]侯敢,黄迪南,祝其锋.猪荃多糖对小鼠腹腔巨噬细胞一氧化氮生成的影响及其机理[J].中国老年学杂志,2000,20(4):233-235.
[87]侯敢,黄迪南,杨明,等.芦荟多糖对小鼠腹腔巨噬细胞NO生成和iNOS活性的影响[J].湖南中医学院学报,2006,26(3):20-23.
[88]陈伟,林新华,黄丽英,等.库拉索芦荟多糖对小鼠腹腔巨噬细胞一氧化氮生成的影响[J].中国现代应用药学杂志,2006,23(1):17-20.
[89]Alain T,Ziad M.Cytokines in atherosclerosis:pathogenic and regulatory pathways[J].Physiological Reviews,2006,86(2):515-581.
[90]Wu M J,Weng C Y,Wang L,et al.Immunomodulatory mechanism of the aqueous extract of sword brake fern(Pteris ensiformis Burm.)[J].Journal of Ethnopharmacology,2005,98(1-2):73-81.
[91]Luk J M,Lai W,Tam P,et al.Suppression of cytokine production and cell adhesion molecule expression in human monocytic cell line THP-1 by Tripterygium wilfordii polysaccharide moiety [J].Life Science,2000,67:155-63.
[92]Zhang Y,Kiyohara H,Matsumoto T,et al.Fraction-ation and chemical properties of immunomodulating polysaccharides from roots of Dipsacus asperoides[J].Planta,Medica 1997,63:393-399.
[93]Egger S F,Brown G S,Kelsey L S,et al.Hematopoietic augmentation by a β-(1-4)-linked mannan[J].Cancer Immunology,Immunotherapy,1996,43:195-205.
[94]Chen Y J,Shiao M S,Lee S S,et al.Effect of Cordyceps sinensis on the proliferation and differentiation of human leukemic U937 cells[J].Life Science,1997,60:2349-2359.
[95]江振友,林晨,刘小澄,等.灵芝多糖对小鼠体液免疫功能的影响[J].暨南大学学报(医学版),2003,24(2):51-53.
[96]Nair P K,Rodriguez S,Ramachandran R,et al.Immune stimulating properties of a novel polysaccharide from the medicinal plant Tinospora cordifolialnt[J].Immunopharmacol,2004,4(13):1645-1659.
[97]Gangloff S C,Zahringer U,Blondin C,et al.Influence of CD14 on ligand interactions between lipopolysaccharide and its receptor complex[J].Journal of immunology,2005,175(6):3940-3945.
[98]Lacchini A H,Davies A J,Machintosh D,et al.β-1,3-glucan modulates PKC signalling in Lymnaea stagnalis defence cells:a role for PKC in H2O2 production and downstream ERK activation[J].Journal of Experimental Biology,2006,209(24):4829-4840.
[99]孙春艳,赵伯涛,郁志芳,等.芦笋的化学成分及药理作用研究进展[J].中国野生植物资源,2004,23(5):1-5.
[100]Yoon Y D,Kang J S,Han S B,et al.Activation of mitogen-activated protein kinases and AP-1 by polysaccharide isolated from the radix of Platycodon grandiflorum in RAW 264.7 cells [J].International Immunopharmacology,2 2004,4(12):1477-1487.
[101]Zughaier S M,Zimmer S M,Datta A,et al.Differential induction of the toll-like receptor 4-MyD88-dependent and -independent signaling pathways by endotoxins[J].Infect Immunology,2005,73(5):2940-2950.
[102]Nakamura T,Suzuki H,Wada Y,et al.Fucoidan induces nitric oxide production via p38mitogen-activated protein kinase and NF-κB-dependent signaling pathways through macrophage scavenger receptors[J].Biochemical and Biophysical Research Communications,2006,343(1):286-294.
[103]Lim T S,Na K,Choi E M,et al.Immunomodulating activities of polysaccharides isolated from Panax ginseng[J].Journal of Medicinal Food,2004,7(1):1-6.
[104]Gordon S.Pattern recognition receptors:doubling up for the innate immune responses[J].Cell,2002,111(7):927-930.
[105]Jeon Y J,Kim H M.Experimental evidences and signal transduction pathways involved in the activation of NF-kappa B/Rel by angelan in murine macrophages[J].International Immunopharmacology,2001,1(7):1331-1339.
[106]Han S B,Park S H,Lee K H,et al.Polysaccharide isolated from the radix of Platycodon grandiflorum selectively activates B cells and macrophages but not T cells[J].International Immunopharmacology,2001,1(11):1969-1978.
[107]Li S G,Zhang Y Q,Zhao J X.Preparation and suppressive effect of astragalus polysaccharide in glornerulonephritis rats[J].International Immunopharmacology.2007,7(1):23-28.
[108]Lee K Y,Jeon Y J.Macrophage activation by polysaccharide isolated from Astragalus membranaceus[J].International Immunopharmacology,2005.5(7-8):1225-1233.
[109]Jeon Y J,Han S B,Ann K S.Activation of NF-κB/Rel in angelan-stimulated macrophages [J].Immunopharmacology,1999,43(1):1-9.
[110]Escribano J,Az-Guerra M J,Riese H H,et al.In vitro activation of macrophages by a novel proteoglycan isolated from corms of Crocus sativus L[J].Cancer Letter.1999,144(1):107-114.
[111]Hsu M J,Lee S S,Lee S T,et al.Signaling mechanisms of enhanced neutrophil phagocytosis and chemotaxis by the polysaccharide purified from Ganoderma lucidum[J].British Journal of Pharmacology,2003,139(2):289-298.
[112]Zuo J,Jin L,Lv J,et al.Induced NO production and iNOS mRNA expression of macrophage by polysaccharide from Paecilomyces tenuipes[J].Chinese Journal of Biochemical Pharmaceutics,2006,27(4):205-208.
[113]Zhang G L,Wang Y H,Ni W,et al.Hepatoprotective liver injury in mice[J].World Journal of Gastroenterology,2002,8(4):728-733.
[114]Zhang B Q,Hu S J,Qiu L H.Effects of Astragalus membranaceus and its main components on the acute phase endothelial dysfunction induced by homocysteine[J].Vascular Pharmacology,2007,46(4):278-285.
[115]候宽昭.中国种子植物科属辞典[M].北京:科学出版社,1982.
[116]郑太坤.中国车前草的形态鉴定[J].中国中药杂志,1990,7:6.
[117]郑太坤.中药车前的研究概况[J].辽宁中医杂志,1985,(8):41.
[118]中华人民共和国卫生部药典委员会.中华人民共和国药典(一部)[M].北京:人民卫生出版社,1990.
[119]王东.车前子多糖的研究[D].辽宁中医学院,2003.
[120]Masashi T,Maemi Y,Kazuyo 1.Plant mucilages.ⅩⅪⅩ.Isolation and characterization of a mucous polysaccharide,"Piantago-mucilage A," from the seeds of Plantago major var.asiatica [J].Chemical & Pharmaceutical Bulletin,1981,29(10):2877-2884.
[121]SamuelsenA B,Lund I,Djahromi J M,et al.Structural features and anti-complementary activity of some heteroxylan polysaccharide fractions from the seeds of Plantago major L[J].Carbohydrate Polymers,1999,38:133-143.
[122]Miyase T,Ishino M,Akahori C,et al.Phenylethanoid Glycosides from Plantago asiatica[J].Phytochemistry,1991,30(6):2015-2018.
[123]Nishibe S,Kodama A,Noguchi Y,et al.Phenolic compounds from seeds of Plantago ovata and P.psyllium[J].Natural Medicines(Tokyo,Japan),2001,55(5):258-261.
[124]Li L,Tsao R,Liu Z Y,et al.Isolation and purification of acteoside and isoacteoside from Plantago psyllium L.by high-speed counter-current chromatography[J].Journal of Chromatography A,2005,1063:161-169.
[125]高明哲,张惠,林立,等.车前子苷的提取分离[J].辽宁中医学院学报,2003,5(2):157.
[126]Patel R B,Rana N G,Patel M R,et al.Chromatographic screening of proteins of seeds of Plantago ovata Forsk[J].Indian Drugs Pharmacology.,1981,16(1):3-5.
[127]付志红.车前子营养与活性成分及其保健功能研究[D].南昌大学,2005.
[128]Rilka T,Ljubka E,Nedjalka H,et al.Iridoid patterns of genus Plantago L.and their systematic significance[J].Journal of Biosciences,2002,57:42-50.
[129]Marteau P,Flourie B,Cherbut C,et al.Digestibility and bulking effect of ispaghula husks in healthy humans[J].Gut,1994,35(12):1747-1752.
[130]Marlett J A,Kajs T M,Fischer M H.An unfermented gel component of psyllium seed husk promotes laxation as a lubricant in humans[J].The American Journal Of Clinical Nutrition,2000,72(3):784-789.
[131]Marlett J A,Fischer M H.A poorly fermented gel from psyllium seed husk increases excreta moisture and bile acid excretion in rats[J].Journal of Nutrition,2002,132(9):2638-2643.
[132]Marlett J A,Fischer M H.The active fraction of psyllium seed husk[J].The Proceedings of The Nutrition Society,2003,62(1):207-209
[133]Edwards C A,Bowen J,Brydon W G,et al.The effects of ispaghula on rat caecal fermentation and stool output[J].British Journal of Nutrition,1992,68(2):473-482
[134]张今强.车前子多糖微丸缓泻制剂的研究[D].辽宁中医学院,2005.
[135]吴光杰,田颖刚,谢明勇,等.车前子多糖对便秘模型小鼠通便作用的研究[J].食品科学,2007,28(10):514-516.
[136]Karen W,Peter B.Reduction of glycenic and lipid levels in db/db diabetic mice by Psyilium plant fiber[J].Diabetes,1989,38(12):1528-1533.
[137]Frati Munari A C,Benitez Pinto W,Raul Ariza Andraca C,et al.Lowering glycemic index of food by acarbose and Plantago psyllium mucilage[J].Archives Of Medical Research,1998,29(2):137-141.
[138]Narayan R.Meet Psyllium:A fiber product with potential cardioprotective Effects[J].Nutrition Noteworthy,2005,7,Article 3.
[139]栗艳彬.车前子胶调血脂及降血糖作用的实验研究[D].辽宁中医学院,2004.
[140]Tomoda M,Takada K,Shimizu N,et al.Reticuloendothelial system-potentiating and alkaline phosphatase-inducing activities of Plantago-mucilage A,the main mucilaget from the seed of Plantago asiatica,and its five modification products[J].Chemical & Pharmaceutical Bulletin,1991,39(8):2068-2071.
[141]谢小梅,付志红,谢明勇,等.精制车前子多糖对小鼠免疫功能的影响[J].第三届全国中医药免疫学术研讨会论文汇编.中国,湖南.2006;18-22.
[142]张振秋,李锋,孙兆妹,等.车前子的药效学研究[J].中药材,1996,19(2):87-89.
[143]Deters A M,Schroder K R,Smiatek T,et.al.Ispaghula(Plantago ovata) seed husk polysaccharides promote proliferation of human epithelial cells(skin keratinocytes and fibroblasts)via enhanced growth factor receptors and energy production[J].Planta Medical,2005,71:33-39.
[144]Westerhof W,Das P K,Middelkoop E,et al.Mucopolysaccharides from psyllium involved in wound heating(Abstract)[J].Drugs under Experimental and Clinic Research,2001,27(5-6):165-175.
[145]韩强,林惠芬,朱玲莉.几种中药提取物对酪氨酸酶活性的抑制[J].香料香精化妆品,1998,4:22-23.
[146]Shortman K,Caux c.dendritic cells development:multiple pathways to nature's adjuvants [J].Stem Cells.1997,15(6):409-419.
[147]Korting H C,Herzinger T.Hartinger A,et al.Discrimination of the irritancy potential of surfactants in vitro by two cytotoxicity assays using normal human keratinocytes,HaCaT cells and 3T3 mouse fibroblasts:correlation with in vivo data from a soap chamber assay[J].Journal Dermatological Science,1994,119-129.
[148]张卓然.培养细胞学与细胞培养技术[M].上海:上海科学技术出版社,2004.
[149]董伟华,郑智敏,刘桂亭.中药大黄、槐米、红花提取物的致突变作用[J].河南医科大学学报,1991,2(64):330-333.
[150]张建清,苏城玉,权玉玲,等.四种补益中药的急性、毒性利致突变性研究[J].现代预防医学,1999,2(611:26-28.
[151]谢建华.青钱柳多糖的分离纯化与结构解析及其生物活性研究[D].南昌大学,2007.
[152]Nie S P,Xie M Y,Fu Z H,et al.Study on the purification and chemical compositions of tea glycoprotein[J].Carbohydrate Polymers,2008,71:626-633.
[153]殷军艺,聂少平,付志红,等.大粒车前子多糖分离、纯化及单糖组成分析[J].食品科学,2008,29(09):529-532.
[154]周超.车前子多糖分离、纯化及功能特性研究[D].南昌大学,2007.
[155]万茵.车前子多糖、黄酮和苯乙醇苷类的纯化、结构解析及其活性功能研究[D].南昌大学,2007.
[156]戴建国,李超,黄培林.高密度壳聚糖对L-929细胞的毒性研究[J].东南大学学报(医学版),2008,27(1):54-57.
[157]翟中和,王喜忠,丁明孝.细胞生物学[M].北京:高等教育出版社,2000.
[158]Inaba k,Inaba M,Romani N,et al.Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulation factor[J].Journal of Experimental Medicine,1992,176:1693-1702.
[159]夏俊波,吴奎,孙鲲,等.小鼠骨髓来源的树突状细胞体外扩增与鉴定[J].第三军医大学学报,2005,27(10):942-944.
[160]Basak S K,Harui A,Stolina M,et al.Increased dendritic cell number and function folowing continuous in vivio infusion of granulocyte macrophage-colony-stimulation factor and interleukin-4[J].Blood,2002,99(8):2869-2879.
[161]罗建飞,陈必成,陈忠华.小鼠骨髓来源树突状细胞的分离与扩增培养[J].微循环学杂志,2005,15(1):29-31.
[162]Markowicz S,Engleman E G.Granulocyte-macrophage colony-stimulating factor promotes differentiation and survival of human perpheral blood dendritic cells in vivo[J].Journal of Clinical Investigation,1990,85:955-961.
[163]Gang M Z,Ying K.Cytokines in the generation and maturation of dendritic cells:recent advances[J].European Cytokine Network,2002;13(2):186-199.
[164]Aerts-Toegaert C,Heirman C,Tuyaerts S.CD83 expression on dendritic cells and T cells:correlation with effective immune responses[J].European Journal of Immunology,2007,37:686-695.
[165]Lutz M B,Suri R M,Niimi M,et al.Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo[J].European Journal of Immunology,2000,30(7):1813-1822.
[166]Cella M,Sallusto F,Lanzavecchia A,et al.Origin,maturation and antigen presenting function of dendritic cells[J].Current opinion in Immunology,1997,9(1):10-16.
[167]黄贝贝,肖凤仪,张文平,等.青钱柳对小鼠免疫功能的影响[J].江西中医学院学报,2004,16(5):59-60.
[168]汪东风,李俊,王常红,等.茶叶多糖的组成及免疫活性研究[J].茶叶科学,2000,20(1):45-50.
[169]R M Steinman.The dendritic cell system and its role in immunogenicity[J].Annual Review of Immunology,1991,9:271-296.
[170]Lin Y L,Liang Y,Lee S S,et al.Polysaccharide purified from Ganoderma lucidum induced activation and maturation of human monocyte-derived dendritic cells by the NF-κB and p38mitogen-activated protein kinase-pathways[J].Journal of Leukocyte Biology Volume,2005,78:533-543.
[171]Roake J A,Rao A S,Morris P J,et al.Dendritic cell loss from nonlymphoid tissues after systemic administration of lipopolysaccharide,tumor necrosis factor,and interleukin 1[J].Journal of Experimental Medicine,1995,181:2237-2247.
[172]Sallusto F,Celia M,Danieli C,et al.Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class Ⅱcompartment:downregulation by cytokines and bacterial products[J].Journal of Experimental Medicine,1995,182:389-400.
[173]O'Sullivan B J,Thomas R.CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-κB[J].Journal of Immunology,2002,168:5491-5498.
[174]Xie J,Qian J,Wang S,et al.Novel and detrimental effects of lipopolysaccharide on in vitro generation of immature dendritic cells:involvement of mitogen-activated protein kinase p38[J].Journal of Immunology,2003,171:4792-4800.
[175]Omarsdottir S,Olafsdottir E S,Freysdottir J,et al.Immunomodulating effects of lichen-derived polysaccharides on monocyte-derived dendritic cells[J].International Immunopharmacology,2006,6:1642-1650.
[176]Kim J Y,Yoon Y D,Ahn J M.Angelan isolated from Angelica gigas Nakai induces dendritic cell maturation through toll-like receptor 4[J].International Immunopharmacology,2007,7:78-87.
[177]Kim G Y,H Cho,Ahn S C,et al.Resveratrol inhibits phenotypic and functional maturation of murine bone marrow-derived dendritic cells[J].International Immunopharmacology,2004,4:245-253.
[178]Liu Q.Chen T,Chen G,et al.Immunosuppressant triptolide inhibits dendritic cell-mediated chemoattraction of neutrophils and T cells through inhibiting Stat3 phospborylation and NF-κB activation[J].Biochemical and Biophysical Research Communications,2006,345:1122-1130.
[179]Zhou L J,Tedder T F.Human blood dendritic cells selectively express CD83,a member of the immunoglobulin superfamily[J].Journal of Immunology,1995,154(8):3821-3835.
[180]Adams S,O'Neill D W,Bhardwaj N.Recent advances in dendritic cell biology[J].Journal of Clinical Immunology,2005,25(3):177-188.
[181] Wu L, Vremec D, Ardavin C, et al. Mouse thymus dendritic cells: kinetics of development and changes in surface markers during maturation [J]. European Journal of Immunology, 1995,25(2): 418-425.
[182] Pieters J. MHC class 11 restricted antigen presentation [J]. Current Opinion in Immunology,1997, 9(1):89-96.
[183] Lu L, Qian S, Hershberger P A, et al. Fas ligand (CD95L) and B7 expression on dendritic cells provide counter-regulatory signals for T cell survival and proliferation [J]. Journal of Immunology, 1997, 158(12): 56-76.
[184] Koch R. High level 1L-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10 [J]. Journal of Experimental Medicine, 1996, 184: 741-747.
[185] Moore K W, de Waal Malefyt R, Coffman R L, et al. Interleukin-10 and interleukin-10 receptor [J].Annual Review Immunology, 2001, 19 : 683—765.
[186] Bogdan C. Nitric oxide and the immune response [J]. Nature Immunology, 2001, 2: 907—916.
[187] Philip S. IL-12: initiation cytokine for cell-mediated immunity [J]. Science, 1993, 260:496-497.
[188] Macatonia S E, Hosken N A, Litton M, et al. Dendritic cells produce IL-12 and direct the decelopment of Thl cells from naive CD4+ T cells [J]. Journal of Immunology, 1995, 154:5071-5079.
[189] Perona-Wright G, Jenkins S J, Crawford A, et al. Distinct sources and targets of IL-10 during dendritic cell-driven Thl and Th2 responses in vivo[J]. European Journal of Immunology,2006,36(9):2367-2375.
[190] Ebner S, Ratzinger G, Krosbacher B, et al. Production of IL-12 by human monocyte-derived dendritic cells is optimal when the stimulus is given at the onset of maturation, and is further enhanced by IL-4 [J]. The Journal of Immunology, 2001, 166: 633—641.
[191] Lamont A G, Adorini L. IL-12, a key cytokine in immune regulation [J]. Immunol Today, 1996, 17(5): 214-217.
[192] Austyn J M, Kupiec-Weglinski J W, Hankins D F, et al. Migration patterns of dendritic cells in the mouse. Homing to T cell-dependent areas of spleen, and binding within marginal zone [J].Journal of Experimental Medicine, 1998, 67: 646—651.
Austyn J M. Migration patterns of dendritic leukocytes [J]. Res. Immunol, 1 989, 140: 898—902.
[193] Shortman K, Liu Y J. Mouse and human dendritic cell subtypes [J]. Nature Reviews Immunology. 2002, 2(3) :151 —161.
[194] Wilson N S, Villadangos J A. Regulation of antigen presentation and cross-presentation in the dendritic cell network:facts.hypothesis,and immunological implications [J]. Advances in immunology, 2005, 86: 241-305.
[195] Guermonprez P, Valladeau J, Zitvogel L, et al. Antigen presentation and T cell stimulation by dendritic cells [J]. Annual Review of Immunology, 2002, 20: 621 —667.
[196]Thery C,Amigorena S.The cell biology of antigen presentation in dendritic cells[J].Current Opinion in Immunology,2001,13(1):45-51.
[197]Jonathan M,Austyn,Ralph M,et al.Dendritic cells initiate a two-stage mechanism for T iymphyocyte proliferation[J].The Journal of Experimental Medicine,1983,157(4):1101-1115.
[198]Dieu-Nosjean M C,Vicari A,Lebecque S,et al.Reuglation of dendritic cell trafficking:a proeess that involves the participation of selective chemokines[J].Journal of Leukocyte Biology,1999,66:252-262.
[199]Setinman R M.The dendritic cell system and its role in immunogencity[J]Annual Review of Immunology,1991,9:271-296.
[200]Sozzani S,Allvaena,P Vecchi A,et al.Chemokines and dendritic cell traffic[J].Journal of Clinical Immunology,2000,20:151-160.
[201]Sallusto F,Palermo B,Lenig D,et al.Distinct Patterns and kinetics of chemokine production regulate dendritic cell function[J].European Journal of Immunology,1999,29(5):1617-1625.
[202]Caux C,Ait-Yahia S,Chemin K,et al.Dendritic cell biology and regulation of dendritic cell trafficking by chemokines[J].Springer Semin Immunopathol,2000,22(4):345-369.
[203]Forster R,Schubel A,Beritefld D,et.al.CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoidorgans[J].Cell,1999,99:23-33.
[204]张卓然.培养细胞学与细胞培养技术[M].上海:上海科学技术出版社,2004.
[205]Dieu M C,Vanbervlietn B,Vieari A,et al.Seleetive recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anato-mic sites[J].The Journal of Experimental Medicine.1998,188(2):373-386.
[206]Cyster J G.Chemokines and the homing of dendritic cells to theTcell areas of lymphoid organ[J].The Journal of Experimental Medicine,1999,189(3):447-450.
[207]Hirao M,Onai N,Hiroishi K,et al.CC chemokine receptor-7 on dendritic cells is induced after interaetion with apoptotic tumor cells:critical role in migration from the tumor site to draining lymph nodes[J].Cancer Research,2000,60(8):2209-2217.
[208]Sato K,Kawasaki H,Nagayama H,et al.Signaling events following chemokine receptor ligation in human dendrtic cells atdiffrent developmenta lstages[J].International Immunology,2001,13(2):167-179.
[209]长琦,许有玲,等.抗肿瘤植物药及其有效成分[M].北京:中国中医药出版社,1997.
[210]Ishida T,Oyma T,Cabone D P,et al.Defective function of Langerhans cells in tumor-bearing animals is the result of defective maturation from hemopoietic progenitors [J].Journal of Immunology,1998,161(9):4842-4851.
[211]Dmitry I,Frank C,David P,et al.Dendritic cells in antitumor immune responses defective antigen presentation in tumor-bearing hosts[J].Cell Immunology,1996,170(1):101-103.
[212]何彦丽,苏俊芳.中药多糖抗肿瘤免疫研究的新视角——对树突状细胞的影响[J].中药药理与临床,2002,18(4):47-49.
[213]Matsumoto M,Fu Y X,Molina H,et al.Distinct roles of lymphotoxin and the typel tumor necrosis factor(TNF) reeptor in the catablishment of folliuclar dendrtic cells from non-bone marrow dendritic cells[J].The Journal of Experimental Medicine,1997,186:1997-2004.
[214]Chiang L C,Chiang W,Chang M Y,et al.In vitro cytotoxic,antiviral and immunomodulatory effects of Plantago major and Plantago asiatica[J].American Journal of Chinese Medicine,2003,31(2):225-234.
[215]Figdor C G,de Vriesl J,Lesterhuis W J,et al.Dendritic cell Immuno-therapy:mapping the way[J].Nature Medicine,2004,10(5):475-480.
[216]Jeofford M,Marskovsky E,Cebon J,et al.The use of dendritic cells in cancer therapy[J].The Lancet Oncoiogy,2001,2(6):343-353.
[217]Nair S K,Snyder D,Rouse B T,et al.Regression of tumors in mice vaccinated with professional antigen-presenting cells pulsed with tumor extracts[J].International Journal of Cancer,1997,70:706-715.
[218]Hattori TS,Komatsu N,Shichijo S,et al.Protein-bound polysaccharide K induced apoptosis of the human Burkitt lymphoma cell line,Namalwa[J].Biomedicine &Pharmacotherapy 58(2004):226-230.
[219]De V S,Arredouani M,Ade M,et al.Uptake and presentation of malignant glioma tumor cell lysates by monocyte-derived dendritic cells[J].Cancer Immunology lmmunotherapy,2005,54(4):372-382.
[220]Kumamoto T,Morita A,Takashima A.Recent advances in dendritic cell vaccines for cancer treatment[J].the Journal of Dermatology,2001,28(11):658-662.
[221]Samuelsen A B.The traditional uses,chemical constituents and biological antivities of Plantago major L.[J].Journal of Ethnopharmacology,2000,71:1-21.
[222]Ji Z,Tang Q,Zhang J,et al.Immunomodulation of RAW264.7 macrophages by GLIS,a proteopolysaccha-ride from Ganoderma lucidum[J].Journal of Ethnopharmacology,2007,112(3):445-450.
[223]游育红,林志彬.灵芝多糖肽对小鼠腹腔巨噬细胞一氧化氮产生的影响[J].中国药理学通报,2004,20(12):1398-1401.
[224]李俊,黄艳,廖日权,等.罗汉果多糖对小鼠免疫功能的影响[J].中国药理学通报,2008,24(9):1237-1240.
[225]Shishodia S,Aggarwal B B.Nuclear factor-kappaB Activation:A question of life or death [J].The Journal of Steroid Biochemistry and Molecular Biology,2002,35(1):28-34.
[226]Zhang L,Tizard I R.Activation of a mouse macrophage cell line by acemannan:the major carbohydrate fraction from Aloe vera gel[J].Immunopharmacology,1996,35(2):119-128.
[227]Karaca K,Sharma J M,Nordgren R.Nitric oxide production by chicken macrophages activated by acemannan,a complex carbohydrate extracted from Aloe vera[J].International Journal of Immunopharmacology,1995,17(3):183-188.