臺灣博碩士論文加值系統

靈芝在分類學上屬於擔子菌綱，為典型的(中)藥用真菌代表。針對多醣體之生物活性，過去的研究發現，病原性微生物(例如: Haemophilus influenza type B 和 Streptococcus pneumonia)的細胞壁多醣屬於免疫原，其抗原主要是結構主要是由特定寡糖單元重覆性排列所組成之高分子，臨床上此類抗原可引起T細胞非依賴型之抗體免疫反應。自古以來，靈芝常被當作免疫調節的保健品或抗癌的輔助品，儘管。有效成分與抗癌的機轉仍在持續研究當中，我們推測靈芝所富含的多醣體成分與活化人體免疫能力之功效有關。從醣類生物學的角度，醣鏈末端具有岩藻糖或唾液酸修飾屬於重要的腫瘤相關醣類抗原之醣化特徵，根據生物表面獨特的醣類結構作為抗原標靶，發展以碳水化合物為基礎的醣類接合型疫苗，已證實可有效的治療癌症與感染症。本研究為探討含岩藻糖的靈芝多醣體是否具有活化抗體生成協同抗腫瘤作用，我們將靈芝多醣體萃取物 (命名為F3 and F3 subfraction, FMS) 採取腹腔注射法免疫小鼠，在移植鼠源性肺腺癌細胞(LLC1)誘發小鼠原位腫瘤的動物模式中，活體試驗結果顯示，受免疫的腫瘤小鼠可延緩癌細胞的生長與降低癌症相關的發炎物質分泌；在探討抗腫瘤的機制中，我們首先發現受免疫的健康小鼠之免疫血清(in vitro)具毒殺癌細胞的活性，進一步利用全醣晶片高通量篩檢抗體的特異性，證實免疫血清中IgM抗體所辨識之醣分子與腫瘤醣類抗原相關，其抗原決定基最小單元是Fuca1-2Gab1-3GalNAc-R；例如:著名的六分子醣脂質Globo H (Fucα1-2Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glcβ)，已知大量表現於乳癌、乳癌幹細胞、肺癌、卵巢癌等多種癌細胞組織中。儘管採用腹腔注射多醣體，活化小鼠體內的免疫系統之運作機制尚未通盤釐清，有趣的是我們發現受免疫的健康小鼠腹腔內的B1 B-細胞數目顯著增加，在體外(in vitro)實驗中，我們更進一步證明靈芝多醣體可刺激受免疫小鼠的B1 B-細胞之分化與抗體分泌，種種證據符合過去的研究報導推測，此亞型B-細胞的活化與其所特有辨識多醣分子之能力有關。本研究中我們除了證明免疫血清的抗腫瘤活性與癌細胞表面Globo H醣類抗原的表現有關，更進一步，我們證實了靈芝多醣體的岩藻糖修飾會直接影響小鼠抗腫瘤抗體的生成，因此，在醣類結構的鑑定上，我們首先著眼於醣鏈末端具岩藻糖的鍵結方式與醣鏈定序。配合使用岩藻糖水解酵素、化學性酸水解與化學衍生化的方式調理靈芝多醣體 (FMS)，綜合各式質譜儀測定的結果，我們推測其主幹由1,4連結的甘露聚糖和1,6連結的半乳聚糖兩種多醣聚合物構成，其末端含岩藻糖的鍵結特徵為Fucα1-2Gal，Fucα1-3/4Man，Fucα1-4Xyl 和 Fucα1-2Fuc；因此，我們預期此分析結果所提供的醣鏈資訊，將有利於爾後含岩藻醣的靈芝多醣體之結構鑑定。總結，本研究提出一可行的天然物多醣體之活性篩選模式，不僅突顯全醣晶片的應用價值，我們也闡述了靈芝多醣體誘發特異性抗醣類抗體具抗腫瘤的免疫機制。

Ganoderma lucidum (Reishi), a mushroom commonly used as a Chinese herb medicine, contains complex polysaccharides that have been used as an antitumor supplement with a rarely understood mechanism of immune response. Here we demonstrated that the mice immunized with a fucose-enriched Reishi polysaccharide fraction (designated as FMS) were able to induce antibodies against murine Lewis lung carcinoma (LLC1) cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular monocyte chemoattractant protein-1, MCP-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycan antigens, with the antigenic structure located in the non-reducing termini, i.e., Fuca1-2Galb1-3GalNAc-R(where R represents reducing end), typically found in Globo H and related tumor antigens. Although the composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-a-galactan, a fucose-dependent nanoLC-tandem MS analysis uncovered the Fuca1-2Gal, Fuca1-3/4Man, Fuca1-4Xyl and Fuca1-2Fuc linkages in the non-reducing termini of the FMS glycans, underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycan antigens. In summary, our development of carbohydrate immunomodulation-based therapy, which successfully correlated the high-throughput glycan microarray analysis with detailed structural analyses of glycan antigens, should be applicable to other medicinal polysaccharides.

1.Introduction(1)
1.1 Basidiomycetes polysaccharide(1)
1.2 Polysaccharide-based vaccine(3)
1.3 Carbohydrate mimicry and autoantibody induction(8)
1.4 Specific aims(12)
2. Methods and Materials(15)
2.1 Purification of a fucose-enriched F3 polysaccharides fraction, FMS(16)
2.2 Characterization of FMS(16)
2.3 Glycan binding analysis of serum IgM antibodies(17)
2.4 Hydrolysis reaction(18)
2.5 Fucose-based nanoLC-tandem MS analysis(19)
2.6 Mice immunization schedule and the lung tumor model(20)
2.7 Serological analysis of serum antibodies(21)
2.8 Fucose-specific plant lectins binding(22)
2.9 Cell cultures and FACS analysis(23)
2.10 Mouse cytokine/chemokine detection(24)
2.11 Antibody-mediated complement-dependent cytotoxicity (CDC) analysis(24)
2.12 Binding competition assay(25)
3. Results and Discussions(26)
3.1 Antitumor activity of F3(26)
3.2 Characterization of fucose-enriched polysaccharides,FMS (28)
3.3 The glycan-binding specificity of the sera IgM antibodies obtained from FMS- treated mice(30)
3.4 FMS possesses antitumor efficacy in vivo(32)
3.5 Terminal fucose of FMS is important for the antibody-mediated antitumor efficacy(33)
3.6 Immunization of FMS stimulates B1 B-cell activation(37)
3.7 Identification of fucosyl glycan moieties of FMS by MS-based approach(39)
3.8 Summary(41)
4. Figures(43)
1.1 Experimental design(13)
4.1 Antitumor effects of F3(43)
4.2 Glycan microarray analysis of F3-treated mice sera(45)
4.3 List of 60 synthetic glycan structures of the fabricated glycan chips(46)
4.4 Representative MALLS profiles of polysaccharides from our Reishi extract(47)
4.5 Glycan-binding patterns of the serum IgM antibodies as measured by the CFG glycan microarray(48)
4.6 A spectrum of tumor associated-glycans highly recognized by FMS-induced sera(49)
4.7 Antitumor activities of fucose-enriched F3 polysaccharides, FMS(50)
4.8 Terminal fucose of FMS is important for the antibody-mediated antitumor efficacy(52)
4.9 Terminal fucose of FMS is correlated with specific antiglycan IgM production(54)
4.10 B1 B-cell expansion in the mice immunization with our Reishi polysaccharides, FMS(55)
4.11 (A)Competition assay;(B)MALDI-MS mapping(57)
4.12 Targeted nanoLC-MS/MS glycan sequencing(58)
4.13 Fucosylated epitope linkage determination of permethylated FMS hydrolysate alditols(59)
4.14 Possible structures of FMS(60)
4.15 Fucose-containing Reishi polysaccharides exert antibody-mediated antitumor activity(45)
5.Tables(62)
1.1 Antitumor mushroom polysaccharides(15)
1.2 Examples of streptococcal capsular polysaccharides by exiting or Developmental carbohydrate-based vaccines(15)
5.1 Glycosyl-linkage composition of FMS and DFMS(62)
5.2 List of glycans bound by F3-induced sera IgM as ranked in decreasing order of binding intensities(63)
5.3 Compositional assignments of multiple charged sodiated molecular ions Observed in nanoLC-MS spectra of permethylated FMS hydrolysate alditols(67)
6.References(68)

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