【作者】 韩澄；

【导师】 谢明勇；

【作者基本信息】 南昌大学 ， 营养与食品卫生学， 2010， 硕士

【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。更多还原

【Abstract】 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.更多还原