【作者】 黄丹菲；

【导师】 谢明勇；

【作者基本信息】 南昌大学 ， 食品科学， 2009， 博士

【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。更多还原

【Abstract】 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.更多还原