臺灣博碩士論文加值系統

近年來，植物性多醣被認為是一種有效且低毒性的生物反應修飾劑(biological response modifier)，目前已經有多種植物性多醣被應用在佐劑的開發，本研究利用番石榴和諾麗之葉片分別以水萃、煮萃以及熱乙醇方式萃取植物多醣，經酚-硫酸法(phenol-sulfuric acid method)進行總醣定量，再以MTT assay檢測萃取物之細胞毒性，最後利用卵白蛋白(ovalbumin)為抗原搭配不同多醣萃取物分析所誘發具專一性的細胞性和體液性免疫反應，來評估多醣萃取物的免疫調節及做為佐劑之潛力。在in vitro試驗中，諾麗葉與番石榴葉之多醣萃取物以1mg/ml~2.5mg/ml的劑量刺激巨噬細胞J774A.1皆具有80%的細胞存活率，相較於市售的植物性佐劑Quil A展現了低毒性的特性。以多醣萃取物刺激巨噬細胞，再以real-time PCR偵測IFN-r、IL-4、IL-6、IL-12等細胞激素表現，分析所誘發的免疫路徑。結果顯示番石榴水萃多醣(GWps)、煮萃多醣(GBps)、醇萃多醣(GEps)和諾麗煮萃多醣(NBps)可以同時刺激Th1和Th2細胞激素表現，而諾麗水萃多醣(NWps)以及醇萃多醣(NEps)僅能誘發Th1細胞激素表現。依據總醣產率、細胞毒性以及刺激細胞激素的效力，挑選NWps、NBps、GBps和GEps進行小鼠免疫，Quil A為陽性對照組；PBS為陰性對照組。在in vivo試驗中，刀豆素A (Concanavalin A)和卵白蛋白誘導脾臟細胞增生和血清中對OVA具有專一之IgG1以及IgG2a抗體效價明顯地被增強。在免疫小鼠之脾臟細胞中，諾麗與番石榴葉多醣也明顯誘發Th1 (IL-12及IFN-r)和Th2 (IL-4及IL-6)細胞激素之產生，並且有效提升NF-kB轉錄因子的表現。綜合以上結果，諾麗葉與番石榴葉之多醣萃取物能提升細胞性及體液性免疫，是一個安全且有效之佐劑，具有應用在預防和治療性疫苗之潛力。

In recent years, plant polysaccharides are considered to be an effective and low toxicity of biological response modifiers. Currently, A variety of plant polysaccharides have been used in the development of adjuvant. In this study, the extractions of plant polysaccharides were extracted with water, boiling, hot ethanol from the leaves of guava and noni. By the phenol sulfuric acid method for quantification of total sugar, and then evaluated for its toxicity with MTT assay. Finally, using different polysaccharide extracts with OVA as antigen assess immunemodulatory and adjuvant potential on the specific cellular and humoral immune response. In vitro, noni and guava polysaccharide of leaf extract to 1 mg/ml ~ 2.5 mg/ml doses stimulate macrophage J774A.1 with 80% cell viability. Compared to commercially available plant-derived adjuvant Quil A showed the characteristics of low toxicity. To polysaccharide extract stimulated macrophages and then detection of cytokine expression with real-time PCR such as IFN-r, IL-4, IL-6, IL-12, and analysis of induced immune path. The results showed that guava water extraction (GWps), boiling extraction (GBps), ethanol extraction polysaccharides (GEps) and noni boiled extracts polysaccharide (NBps) can stimulate the expression of Th1 and Th2 cytokines. Noni water extraction (NWps) and ethanol extraction polysaccharides (NEps) can only induce Th1 cytokine expression. Based on total sugar yield, cell toxicity and the effect of cytokine stimulation selection NWps, NBps, GBps and GEps for immune, Quil A as a positive control, PBS as a negative control. In vivo, the Con A and OVA-induced splenocyte proliferation and the serum OVA-specific IgG1 and IgG2a antibody titers in the immunized mice were significantly enhanced. The leaves polysaccharide with Noni and guava also significantly promoted the production of Th1 (IL-12 and IFN-r) and Th2 (IL-4 and IL-6) cytokines and NF-kB transcription factors in splenocytes from the immunized mice. The results indicated that polysaccharide of leaves extract with noni and guava had strong potential to increase both cellular and humoral immune responses, and that is a safe and efficacious adjuvant candidate suitable for a wide spectrum of prophylactic and therapeutic vaccines.

目錄.........................................................................Ⅰ
表目錄.......................................................................Ⅴ
圖目錄.......................................................................Ⅵ
附錄目錄.....................................................................Ⅶ
中文摘要......................................................................1
英文摘要......................................................................2
第一章 前言...................................................................4
1.1 疫苗簡介..................................................................4
1.1.1 疫苗的起源..............................................................4
1.1.2 疫苗的分類與目的........................................................4
1.1.3 疫苗的作用原理..........................................................5
1.2 疫苗佐劑..................................................................5
1.2.1 疫苗佐劑之簡介..........................................................5
1.2.2 疫苗佐劑之作用機制......................................................6
1.2.3 佐劑的理想條件…........................................................7
1.2.4 疫苗佐劑的分類..........................................................8
1.2.5 佐劑發展的阻礙..........................................................8
1.3 植物性多醣................................................................9
1.3.1 植物性多醣的來源與治療潛力..............................................9
1.3.2 植物性多醣刺激巨噬細胞活化.............................................10
1.4 諾麗葉...................................................................11
1.5 番石榴葉.................................................................12
1.6 NF-κB....................................................................12
1.7 Quil A...................................................................13
1.8 細胞激素.................................................................15
1.9 研究目的.................................................................15
第二章 材料與方法............................................................17
2.1 實驗材料...........................................................................17
2.1.1 細胞株.................................................................17
2.1.2 儀器與裝置.............................................................17
2.1.3 藥品與試劑...........................................................................18
2.1.4 植物來源...............................................................19
2.1.5 實驗動物...............................................................19
2.2 實驗方法.................................................................19
2.2.1 樣本前處理.............................................................19
2.2.2 粗萃液製備.............................................................19
2.2.3 多醣製備...............................................................20
2.2.4 總醣定量...............................................................20
2.2.5 蛋白質定量...........................................................................21
2.2.6 細胞培養...............................................................21
2.2.7 細胞存活率測試.........................................................22
2.2.8 刺激 J774A.1 細胞激素表現..............................................22
2.2.9 mRNA 抽取..............................................................22
2.2.10 cDNA 合成.............................................................23
2.2.11 即時定量 Real-time PCR................................................23
2.2.12 免疫動物試驗..........................................................24
2.2.13 抗血清的製備..........................................................24
2.2.14 酵素連結免疫吸附分析..................................................24
2.2.15 血清抗體 IgG 亞型分析.................................................25
2.2.16 免疫後小鼠脾臟細胞之取得..............................................25
2.2.17 脾臟細胞OVA 專一性抗原增生試驗........................................26
2.2.18 免疫後小鼠脾臟細胞之激素表現分析......................................27
2.2.19 免疫後小鼠脾臟細胞之轉錄因子NF-κB 表現分析............................27
2.2.20 統計分析..............................................................27
第三章 結果..................................................................28
3.1 萃取物之蛋白質含量.......................................................28
3.2 萃取物之總醣含量.........................................................28
3.3 萃取物刺激巨噬細胞-細胞存活百分比........................................29
3.4 萃取物刺激巨噬細胞-細胞激素表現分析......................................29
3.5 萃取物刺激巨噬細胞-細胞激素劑量效應表現分析..............................30
3.6 免疫後血清抗體效價.......................................................31
3.7 免疫後IgG 抗體亞型分析(IgG1/IgG2a)……...................................31
3.8 免疫後小鼠脾臟細胞OVA 專一性抗原增生率...................................32
3.9 免疫後小鼠脾臟細胞之細胞激素表現分析.....................................32
3.10 免疫後小鼠脾臟細胞之NF-κB 轉錄因子表現分析..............................33
表...........................................................................34
圖...........................................................................38
第四章 討論..................................................................53
參考文獻.....................................................................58
附錄.........................................................................69

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