【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(guó),作為我國(guó)重要的農(nóng)副產(chǎn)品,大量粗老茶葉以及相關(guān)工藝副產(chǎn)品一直未被很好的綜合利用。大量研究發(fā)現(xiàn)茶葉中的多糖類成分具有增強(qiáng)機(jī)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(qiáng)藥理機(jī)制,本研究建立應(yīng)用小鼠骨髓來源樹突狀細(xì)胞(dendritic cells,DCs)為靶細(xì)胞的免疫活性.的篩選模型,在細(xì)胞水平研究是否具有免疫調(diào)節(jié)功能�，F(xiàn)將本文主要研究結(jié)果概括如下： 1.觀察TGP對(duì)樹突狀細(xì)胞的前體細(xì)胞是否具有毒性。倒置顯微鏡法觀察細(xì)胞形態(tài)；MTT法檢測(cè)對(duì)細(xì)胞增殖率的影響；流式細(xì)胞儀測(cè)定細(xì)胞早期凋亡率。結(jié)果發(fā)現(xiàn)經(jīng)TGP處理后樹突狀細(xì)胞的前體細(xì)胞生長(zhǎng)狀態(tài)良好、細(xì)胞生存率較高,對(duì)樹突狀細(xì)胞的前體細(xì)胞無明顯凋亡影響,實(shí)驗(yàn)結(jié)果表明后續(xù)實(shí)驗(yàn)所采用的TGP無細(xì)胞毒性。 2.建立以樹突狀細(xì)胞為靶細(xì)胞的TGP活性篩選模型。聯(lián)合應(yīng)用rmGM-CSF和rmIL-4誘導(dǎo)分化小鼠骨髓細(xì)胞,并采用MACS免疫磁珠法進(jìn)一步純化。通過細(xì)胞形態(tài)學(xué)及表面分子表達(dá)鑒定所得細(xì)胞,結(jié)果發(fā)現(xiàn)該方法可以獲得大量高純度穩(wěn)定的樹突狀細(xì)胞,可應(yīng)用于后續(xù)實(shí)驗(yàn)研究。 3.應(yīng)用該細(xì)胞活性篩選模型,初步探討TGP的免疫調(diào)節(jié)作用。樹突狀細(xì)胞經(jīng)TGP誘導(dǎo)刺激后的樹突狀細(xì)胞形態(tài)越發(fā)典型;其MHCⅡ、CD40 CD80、CD83和CD86的表達(dá)顯著上調(diào)；其吞噬活性能明顯降低。 4.探討不同實(shí)驗(yàn)劑量茶葉糖蛋白TGP對(duì)樹突狀細(xì)胞分泌功能的影響。結(jié)果發(fā)現(xiàn),TGP能顯著提高樹突狀細(xì)胞的NO分泌量(p0.05),誘導(dǎo)樹突狀細(xì)胞分泌Thl型細(xì)胞因子IL-1β、IL-12p70以及趨化因子RANTES,與此同時(shí)對(duì)Th2型細(xì)胞因子IL-10分泌量抑制顯著(p0.01)。因此TGP可以調(diào)控樹突狀細(xì)胞分泌不同類型細(xì)胞因子和趨化因子,誘導(dǎo)機(jī)體趨向Thl型細(xì)胞免疫應(yīng)答。 5.探討不同劑量茶葉糖蛋白TGP對(duì)樹突狀細(xì)胞活化T淋巴細(xì)胞功能的影響。結(jié)果顯示,在適宜的DCs:T cells比例下,TGP可活化樹突狀細(xì)胞激活由OVA未致敏和致敏淋巴細(xì)胞增殖能力,樹突狀細(xì)胞的抗原提呈與誘導(dǎo)免疫應(yīng)答功能均顯著增強(qiáng)。通過ELISA法檢測(cè)表明TGP可以上調(diào)樹突狀細(xì)胞促進(jìn)T細(xì)胞分泌IL-2、IFN-γ和I[,-10細(xì)胞因子水平。 6.探討不同劑量TGP對(duì)樹突狀細(xì)胞趨化功能的影響。采用半定量PCR法檢測(cè)樹突狀細(xì)胞表面趨化因子受體CCR7和CXCR4的mRNA表達(dá)水平。結(jié)果發(fā)現(xiàn)TGP可顯著提高樹突狀細(xì)胞表面CCR7和CXCR4的mRNA表達(dá),推測(cè)TGP對(duì)樹突狀細(xì)胞成熟的影響可能與趨化因子受體表達(dá)相關(guān)。
[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(hào)：2379725