摘要
茶叶作为世界三大饮料(茶叶、可可、咖啡)之一,最早起源于中国,作为我国重要的农副产品,大量粗老茶叶以及相关工艺副产品一直未被很好的综合利用。大量研究发现茶叶中的多糖类成分具有增强机体免疫力、抗氧化、降血糖、降血脂、降血压、抗艾滋、抗肿瘤、抗辐射等诸多生物活性。
为了探讨茶叶糖蛋自(tea glycoprotein,TGP)的免疫增强药理机制,本研究建立应用小鼠骨髓来源树突状细胞(dendritic cells,DCs)为靶细胞的免疫活性.的筛选模型,在细胞水平研究是否具有免疫调节功能。现将本文主要研究结果概括如下：
1.观察TGP对树突状细胞的前体细胞是否具有毒性。倒置显微镜法观察细胞形态；MTT法检测对细胞增殖率的影响；流式细胞仪测定细胞早期凋亡率。结果发现经TGP处理后树突状细胞的前体细胞生长状态良好、细胞生存率较高,对树突状细胞的前体细胞无明显凋亡影响,实验结果表明后续实验所采用的TGP无细胞毒性。
2.建立以树突状细胞为靶细胞的TGP活性筛选模型。联合应用rmGM-CSF和rmIL-4诱导分化小鼠骨髓细胞,并采用MACS免疫磁珠法进一步纯化。通过细胞形态学及表面分子表达鉴定所得细胞,结果发现该方法可以获得大量高纯度稳定的树突状细胞,可应用于后续实验研究。
3.应用该细胞活性筛选模型,初步探讨TGP的免疫调节作用。树突状细胞经TGP诱导刺激后的树突状细胞形态越发典型;其MHCⅡ、CD40 CD80、CD83和CD86的表达显著上调；其吞噬活性能明显降低。
4.探讨不同实验剂量茶叶糖蛋白TGP对树突状细胞分泌功能的影响。结果发现,TGP能显著提高树突状细胞的NO分泌量(p<0.05),诱导树突状细胞分泌Thl型细胞因子IL-1β、IL-12p70以及趋化因子RANTES,与此同时对Th2型细胞因子IL-10分泌量抑制显著(p<0.01)。因此TGP可以调控树突状细胞分泌不同类型细胞因子和趋化因子,诱导机体趋向Thl型细胞免疫应答。
5.探讨不同剂量茶叶糖蛋白TGP对树突状细胞活化T淋巴细胞功能的影响。结果显示,在适宜的DCs:T cells比例下,TGP可活化树突状细胞激活由OVA未致敏和致敏淋巴细胞增殖能力,树突状细胞的抗原提呈与诱导免疫应答功能均显著增强。通过ELISA法检测表明TGP可以上调树突状细胞促进T细胞分泌IL-2、IFN-γ和I[,-10细胞因子水平。
6.探讨不同剂量TGP对树突状细胞趋化功能的影响。采用半定量PCR法检测树突状细胞表面趋化因子受体CCR7和CXCR4的mRNA表达水平。结果发现TGP可显著提高树突状细胞表面CCR7和CXCR4的mRNA表达,推测TGP对树突状细胞成熟的影响可能与趋化因子受体表达相关。
Tea, Camellia sinensis, as the famous maximum beverage, is one of the first set of medicinal herbs documented in ancient Chinese medicinal literature. Suposedly originated from China, a large number of coarse old tea are unmarketable or overstocked. To date, it has been reported that the polysaccharide components from tea have a broad range of nutritional and therapeutic values in lowering blood sugar, lowering blood pressure, lowering blood lipids, slowing heartbeat, aniblood coagulation, antitumor, anti-HIV, protecting blood phase and enhancing human nonspecific immunity.
We establish a screening model of DCs in order to illustrate the immune effects of tea glycoprotein (TGP). The main results are listed as follows:
1. Investigated the cytotoxity of TGP on bone marrow cells. We found that TGP had not significant inhibition and apoptosis on cell growth and proliferation through cellular morphology, MTT assay and flow cytometry. So it has been proved that TGP can be used in following research for the reason that it has no cytotoxity on bone marrow-derived cells.
2. Established the DCs screening model of immunological activity. Cytokines (rmGM-CSF and rmIL-4) and MACS system were used in our research to induce bone marrow-derived cells to dendritic cells. While the morphology and the expression level of surface molecular of DCs were tested. The results showed that the method of gaining high purity of DCs was established stably, and which could used for the following studies.
3. Investigate the effects of TGP on the phenotypic and functional of DCs. We observed the sheet-like processes typical morphology of DCs stimulated by TGP. We found that TGP could enhance the expression level of MHCⅡ, CD80, CD83, CD86 and CD40 and there was a concentration-dependent relationship. The phagocytosis of DCs was significantly decreased after treated with TGP.
4. Investigated the effect of TGP on the secretion activity of DCs. We found that TGP could enhance the secretion level of nitric oxide and the Th1 related cytokine such as IL-12p70, IL-1βand RANTES but inhibit significantly the IL-10 secretion (p <0.01), besides these effects were dose-depended. These results showed that TGP could impact the secretion activity of DCs.
5. Investigated regulatory effects of TGP on the proliferation of T lymphocyte induced by DCs. The results demonstrated that TGP could enhance antigen presenting ability of DCs to syngeneically naive and allogeneically primed T lymphocytes by using MLR examination. The expression level of IL-2, IFN-γand IL-10 of T lymphocyte was enhanced when the regulatory effects of DCs treated with TGP.
6. Investigated effects of TGP on the expression of CCR7 and CXCR4 mRNA on DCs. The expression level of the chemotactic receptor CCR7 and CXCR4 mRMA was analyzed by using seim-quantitation PCR method. The results show that the mRNA expression of chemotactic factor receptor of DCs could enhanced by TGP.
引文
[1]Steinman RM, Cohn ZA. Identification of novel cell type in peripheral lymphoid organs of mice Morphology, quantification, tissue distribution[J]. J Exp Med,1973,137(5):1142-1162.
[2]Steinman RM. The dendritic cell system and its role in immunogenicity[J]. Annual Review of Immunology.1991,9:271-296.
[3]许亚明,池诏丞,任明,等.树突状细胞的研究进展[J].吉林医学.2008,29(13)：1127-1130.
[4]Schwarz FS. Positive and negative regulation of the myeloid dendritic cell lineage[J]. Journal of Leukocyte Biology.1999,66(2):209-216.
[5]O'sullivan B J, Thomas R. CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-κB[J]. The Journal of Immunology.2002,168: 5491-5498.
[6]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.
[7]Dannull J, Cerny T, Ackermann DK, et al. Current status of dendritic cell-based tumor vaccination[J]. Onkologie.2000,23(6):544-551
[8]Hows JM, Marsh JC, Bradley BA, et al. Human cord blood: a Source of transplantable stem cells[J]. Bone Marrow Transplant.1992,9(suppl1):105-108
[9]OkiM, 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.
[10]Klinkert WE, La Baddie JH, 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.
[11]Rao MS, Mattson MP. Stem cells and aging:expanding the possibilities[J]. Mechanisms of Ageing and Development.2001,122(7):713-734.
[12]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.
[13]Fridenshtein Ala. Stromal bone marrow cells and the hematopoietic microenvironment[J]. Arkh Patol.1982,44(10):3-11.
[14]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.
[15]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.
[16]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.
[17]Smyth M J, Godfrey D I, Crapani J A. A fresh look at tumor immunosurveillance and immunotheraphy [J]. Nature Immunology,2002,2(4):293～299.
[18]Lipscomb M F, Masten B J. Dendritic cells:immune regulators in health and disease [J]. Physiology Review,2002,82(1):97～130.
[19]Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance [J]. Nature Reviews Cancer,2005,5(4):263～274.
[20]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.
[21]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.
[22]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.
[23]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.
[24]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.
[25]蔡鸿恩.中西医结合茶治疗糖尿病初步疗效观察报告[J].茶叶通报.1979,11(2)：58-59.
[26]Ivor E Dreosti. Antioxidant Polyphenols in Tea, Cocoa, and Wine[J]. Nutrition,2000,16: 692-694.
[27]Ramaswamy, R, Ramaswamy, P. Changes in enzyme activities (polyphenol oxidase and phenylalanine ammonialyase) with type of tea leaf and during black tea manufacture and the effect of enzyme supplementation of dhool on black tea quality[J]. Food Chemistry,1998,62: 277-281.
[28]Hidehiko, Y, Takehiko, T. Effect of theanine, γ-glutamylethylamide, on brain monoamines, striatal dopamine release and some kinds of behavior in rats[J]. Nutrition,2000, 16:776-777.
[29]Yasuyuki, S, Tomomi S, Takashi, S. Improvement of idarubicin induced antitumor activity and bone marrow suppression by theanine, a component of tea[J]. Cancer Letters,2000,158: 119-124.
[30]Rycroft, J A, Quinlan, P Q, Lane, J, O'Brien. The effect of caffeine level in tea on psychophysiological responses and mood in human subjects[J]. International Journal of Psychophysiology,1998,30:265.
[31]Bu-Abbas, A, Clifford, M N, Walker, R, Ioannides, C. Contribution of Caffeine and Flavanols in the Induction of Hepatic Phase Ⅱ Activity by Green Tea[J]. Food and Chemical Toxicology,1998,36:617-621.
[32]清水岑夫.探讨茶叶降血糖作用以从茶叶中制取糖尿病的药物[J].国外农学—茶叶.1990,3：38-40.
[33]清水岑夫.从茶叶中制取糖尿病的药物为例探讨茶叶降血糖作用[J].茶[日].1987,(3)：24-27.
[34]张树政.糖生物学与糖生物工程[M].北京：清华大学出版社,2002.
[35]严鸿德.茶叶深加工技术[M].北京：中国轻工业出版社,1998.
[36]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.
[37]Shortman K, Caux c. dendritic cells development:multiple pathways to nature's adjuvants [J].Stem Cells,1997,15(6):409～419.
[38]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.
[39]张建清,苏城玉,权玉玲,等.四种补益中药的急性、毒性和致突变性研究[J].现代预防医学,1999,2(61)：26-28.
[40]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.
[41]聂少平.茶叶糖蛋白的一级精细结构、构象及生物多样性研究[D].南昌大学,2006.
[42]Mi-Hyoung Kim, Hong-Gu Joo. Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cells[J]. Immunology Letters,2008,115:138～143.
[43]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.
[44]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.
[45]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.
[46]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.
[47]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.
[48]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.
[49]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.
[50]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.
[51]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.
[52]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.
[53]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.
[54]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.
[55]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.
[56]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.
[57]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 p38 mitogen-activated protein kinase pathways [J]. Journal of Leukocyte Biology Volume,2005,78: 533～543.
[58]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.
[59]Sallusto F, Cella M, Danieli C, et al. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class Ⅱ compartment:downregulation by cytokines and bacterial products [J]. Journal of Experimental Medicine,1995,182:389～400.
[60]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.
[61]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.
[62]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.
[63]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.
[64]Liu Q, Chen T, Chen G, et al. Immunosuppressant triptolide inhibits dendritic cell-mediated chemoattraction of neutrophils and T cells through inhibiting Stat3 phosphorylation and NF-κB activation [J]. Biochemical and Biophysical Research Communications,2006,345:1122～ 1130.
[65]Adams S, O'Neill D W, Bhardwaj N. Recent advances in dendritic cell biology [J]. Journal of Clinical Immunology,2005,25(3):177～188.
[66]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):41～425.
[67]Pieters J. MHC class Ⅱ restricted antigen presentation [J]. Current Opinion in Immunology, 1997,9(1):89～96.
[68]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.
[69]Koch R. High level IL-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.
[70]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.
[71]Philip S. IL-12:initiation cytokine for cell-mediated immunity [J]. Science,1993,260: 496～497.
[72]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.
[73]Perona-Wright G, Jenkins S J, Crawford A, et al. Distinct sources and targets of IL-10 during dendritic cell-driven Th1 and Th2 responses in vivo[J]. European Journal of Immunology, 2006,36(9):2367-2375.
[74]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.
[75]Lamont A G, Adorini L. IL-12, a key cytokine in immune regulation [J]. Immunol Today,1 996,17(5):214～217.
[76]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.
[77]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.
[78]陈一晴,聂少平,黄丹菲,等.茶叶糖蛋白对小鼠腹腔巨噬细胞分泌功能的影响.Abstracts of Food Summit in China 2009 6th Annual Meeting of CIFST:80.
[79]Mi-Hyoung Kim, Hong-Gu Joo. Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cells [J]. Immunology Letter.2008,115:138-143.
[80]Carla Cristina Squaiella, Renata Zeigler Ananias, Juliana Sekeres Mussalem, et al. In vivo and in vitro effect of killed Propionibacterium acnes and its purified soluble polysaccharide on mouse bone marrow stem cells and dendritic cell differentiation [J]. Immunobiology.2006,211: 105-116.
[81]Jonathan M, Austyn, Ralph M, et al. Dendritic cells initiate a two-stage mechanism for T lymphyocyte proliferation [J]. The Journal of Experimental Medicine,1983,157 (4):1101～ 1115.
[82]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.
[83]Setinman R M. The dendritic cell system and its role in immunogencity [J] Annual Review of Immunology,1991,9:271-296.
[84]Sozzani S, Allvaena, P Vecchi A, et al. Chemokines and dendritic cell traffic [J]. Journal of Clinical Immunology,2000,20:151～160.
[85]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.
[86]Caux C, Ait-Yahia S, Chemin K, et al. Dendritic cell biology and regulation ofdendritic cell trafficking by chemokines [J]. Springer Semin Immunopathol,2000,22(4):345～369.
[87]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.
[88]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.
[89]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.