【摘要】： 茶葉作為世界三大飲料(茶葉、可可、咖啡)之一,最早起源于中國,作為我國重要的農副產品,大量粗老茶葉以及相關工藝副產品一直未被很好的綜合利用。大量研究發(fā)現茶葉中的多糖類成分具有增強機體免疫力、抗氧化、降血糖、降血脂、降血壓、抗艾滋、抗腫瘤、抗輻射等諸多生物活性。 為了探討茶葉糖蛋自(tea glycoprotein,TGP)的免疫增強藥理機制,本研究建立應用小鼠骨髓來源樹突狀細胞(dendritic cells,DCs)為靶細胞的免疫活性.的篩選模型,在細胞水平研究是否具有免疫調節(jié)功能�，F將本文主要研究結果概括如下： 1.觀察TGP對樹突狀細胞的前體細胞是否具有毒性。倒置顯微鏡法觀察細胞形態(tài)；MTT法檢測對細胞增殖率的影響；流式細胞儀測定細胞早期凋亡率。結果發(fā)現經TGP處理后樹突狀細胞的前體細胞生長狀態(tài)良好、細胞生存率較高,對樹突狀細胞的前體細胞無明顯凋亡影響,實驗結果表明后續(xù)實驗所采用的TGP無細胞毒性。 2.建立以樹突狀細胞為靶細胞的TGP活性篩選模型。聯合應用rmGM-CSF和rmIL-4誘導分化小鼠骨髓細胞,并采用MACS免疫磁珠法進一步純化。通過細胞形態(tài)學及表面分子表達鑒定所得細胞,結果發(fā)現該方法可以獲得大量高純度穩(wěn)定的樹突狀細胞,可應用于后續(xù)實驗研究。 3.應用該細胞活性篩選模型,初步探討TGP的免疫調節(jié)作用。樹突狀細胞經TGP誘導刺激后的樹突狀細胞形態(tài)越發(fā)典型;其MHCⅡ、CD40 CD80、CD83和CD86的表達顯著上調；其吞噬活性能明顯降低。 4.探討不同實驗劑量茶葉糖蛋白TGP對樹突狀細胞分泌功能的影響。結果發(fā)現,TGP能顯著提高樹突狀細胞的NO分泌量(p0.05),誘導樹突狀細胞分泌Thl型細胞因子IL-1β、IL-12p70以及趨化因子RANTES,與此同時對Th2型細胞因子IL-10分泌量抑制顯著(p0.01)。因此TGP可以調控樹突狀細胞分泌不同類型細胞因子和趨化因子,誘導機體趨向Thl型細胞免疫應答。 5.探討不同劑量茶葉糖蛋白TGP對樹突狀細胞活化T淋巴細胞功能的影響。結果顯示,在適宜的DCs:T cells比例下,TGP可活化樹突狀細胞激活由OVA未致敏和致敏淋巴細胞增殖能力,樹突狀細胞的抗原提呈與誘導免疫應答功能均顯著增強。通過ELISA法檢測表明TGP可以上調樹突狀細胞促進T細胞分泌IL-2、IFN-γ和I[,-10細胞因子水平。 6.探討不同劑量TGP對樹突狀細胞趨化功能的影響。采用半定量PCR法檢測樹突狀細胞表面趨化因子受體CCR7和CXCR4的mRNA表達水平。結果發(fā)現TGP可顯著提高樹突狀細胞表面CCR7和CXCR4的mRNA表達,推測TGP對樹突狀細胞成熟的影響可能與趨化因子受體表達相關。
[Abstract]:Tea, as one of the three major beverages in the world (tea, cocoa, coffee), originated in China. As an important agricultural by-product in China, a large number of crude old tea and related technological by-products have not been well utilized. A large number of studies have found that the polysaccharides in tea have many biological activities, such as enhancing immunity, antioxidation, lowering blood sugar, lowering blood lipids, lowering blood pressure, anti-AIDS, anti-tumor, anti-radiation and so on. In order to investigate the immunological mechanism of tea sugar egg autogenous (tea glycoprotein,TGP), the immunological activity of mouse bone marrow-derived dendritic cells (dendritic cells,DCs) was established. The screening model was used at the cell level to study whether it had immunomodulatory function. The main results of this paper are summarized as follows: 1. To observe the toxicity of TGP to dendritic cells. The morphology of cells was observed by inverted microscope, the effect of MTT on cell proliferation was detected, and the rate of early apoptosis was measured by flow cytometry. The results showed that the progenitor cells of dendritic cells treated with TGP had a good growth state and a high cell survival rate, and had no obvious effect on the apoptosis of progenitor cells of dendritic cells. The results showed that the TGP used in subsequent experiments had no cytotoxicity. 2. A screening model of TGP activity for dendritic cells was established. Mouse bone marrow cells were induced by rmGM-CSF and rmIL-4 and purified by MACS immunomagnetic beads. The results showed that the method could obtain a large number of high purity and stable dendritic cells, which could be used in the subsequent experimental study. 3. The immunomodulatory effect of TGP was preliminarily studied by using the screening model of cell activity. The morphology of dendritic cells stimulated by TGP was more typical, the expression of MHC 鈪，

本文編號：2379725