請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32262
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 許輔 | |
dc.contributor.author | Ya-Tin Lu | en |
dc.contributor.author | 呂雅婷 | zh_TW |
dc.date.accessioned | 2021-06-13T03:39:32Z | - |
dc.date.available | 2015-08-10 | |
dc.date.copyright | 2011-08-10 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-07-28 | |
dc.identifier.citation | 王伯徹。1990。藥用真菌系列報導(四)茯苓。食品工業。22(5):23-30。
王伯徹。2000。具開發潛力食藥用菇介紹。食品工業。32(5):1-17。 卯曉嵐。1989。中國的食用與藥用大型真菌。微生物學通報。216:290-297。 吳綵籈。2010。台灣金線連免疫調節蛋白基因選殖及表現之研究。國立台灣大學園藝學研究所碩士論文。 宋一洋。2004。茯苓化學成分及藥理活性之研究 茯苓化學成分及藥理活性之研究 (Ⅱ)。國防大學國防醫學院藥學研究所碩士論文。 杜自疆。1980。食用菇栽培技術。豐年社。台北。台灣。198 頁。 林怡君。2002。茯苓化學成分之研究。國防大學國防醫學院藥學研究所碩士論文。 康健雜誌。過敏專刊。2003。 張慧欣。2005。茯苓免疫調節蛋白的純化與生理活性之探討。國立台灣大學園藝學研究所碩士論文。 張慧欣。2009。茯苓免疫調節蛋白活化小鼠腹腔巨噬細胞之訊息傳導路徑及促進 T 細胞活化與第一型 T 輔助細胞免疫反應。國立台灣大學園藝學研究所博士論文。 張樹庭、卯曉嵐。1995。香港蕈菌。中文大學出版社。295-336 頁。 陳健棋。2000。食用菇類在醫藥上的應用。食品工業。32(5):54-66。 黃惠君。2004。食藥用菇的營養與藥用價值。食品工業。36(5):25-32。 楊新美。1988。中國食用菌栽培學。農業出版社。2584 頁。 錢家樂。2004。樟芝免疫調節蛋白基因選殖及表現之研究。國立台灣大學園藝學研究所碩士論文。 顏正華。1997。中藥學 (上冊)。台北。知音出版社。865-867。 Burrows, M.; Assundani, D.; Celis, E.; Tufaro, F.; Tanaka, A.; Bradley, W. G. Oral administration of PPC enhances antigen-specific CD8+ T cell responses while reducing IgE levels in sensitized mice. BMC Complement Altern Med. 2009, 9 49. 39 Chang, H. H.; Yeh, C. H.; Sheu, F. A novel immunomodulatory protein from Poria cocos induces Toll-like receptor 4-dependent activation within mouse peritoneal macrophages. J Agric Food Chem. 2009, 57 (14), 6129-6139. Chang, H.-H.; Hsieh, K.-Y.; Yeh, C.-H.; Tu, Y.-P.; Sheu, F. Oral administration of an Enoki mushroom protein FVE activates innate and adaptive immunity and induces anti-tumor activity against murine hepatocellular carcinoma. International Immunopharmacology. 2010, 10 (2), 239-246. Chen, X. Y.; Xu, X. J.; Zhang, L. N.; Zeng, F. B. Chain conformation and anti-tumor activities of phosphorylated (1 -> 3)-beta-D-glucan from Poria cocos. Carbohyd Polym. 2009, 78 (3), 581-587. Chen, X. Y.; Zhang, L. N.; Cheung, P. C. K. Immunopotentiation and anti-tumor activity of carboxymethylated-sulfated beta-(1 -> 3)-D-glucan from Poria cocos. International Immunopharmacology. 2010, 10 (4), 398-405. El Dine, R. S.; El Halawany, A. M.; Ma, C. M.; Hattori, M. Anti-HIV-1 protease activity of lanostane triterpenes from the vietnamese mushroom Ganoderma colossum. J Nat Prod. 2008, 71 (6), 1022-1026. Frohman, M. A.; Dush, M. K.; Martin, G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988, 85 (23), 8998-9002. Gapter, L.; Wang, Z.; Glinski, J.; Ng, K.-y. Induction of apoptosis in prostate cancer cells by pachymic acid from Poria cocos. Biochemical and Biophysical Research Communications. 2005, 332 (4), 1153-1161. Geethangili, M.; Fang, S. H.; Lai, C. H.; Rao, Y. K.; Lien, H. M.; Tzeng, Y. M. Inhibitory effect of Antrodia camphorata constituents on the Helicobacter pylori-associated gastric inflammation. Food Chem. 2010, 119 (1), 149-153. 40 Giner, E. M.; Manez, S.; Recio, M. C.; Giner, R. M.; Cerda-Nicolas, M.; Rios, J. L. In vivo studies on the anti-inflammatory activity of pachymic and dehydrotumulosic acids. Planta Medica. 2000, 66 (3), 221-227. Giner-Larza, E. M.; Manez, S.; Giner-Pons, R. M.; Carmen Recio, M.; Rios, J. L. On the anti-inflammatory and anti-phospholipase A(2) activity of extracts from lanostane-rich species. J Ethnopharmacol. 2000, 73 (1-2), 61-69. Haak-Frendscho, M.; Kino, K.; Sone, T.; Jardieu, P. Ling Zhi-8: a novel T cell mitogen induces cytokine production and upregulation of ICAM-1 expression. Cell Immunol. 1993, 150 (1), 101-113. Hattori, T.; Hayashi, K.; Nagao, T.; Furuta, K.; Ito, M.; Suzuki, Y. Studies on antinephritic effects of plant components (3): Effect of pachyman, a main component of Poria cocos Wolf on original-type anti-GBM nephritis in rats and its mechanisms. Jpn J Pharmacol. 1992, 59 (1), 89-96. Hsu, H. C.; Hsu, C. I.; Lin, R. H.; Kao, C. L.; Lin, J. Y. Fip-vvo, a new fungal immunomodulatory protein isolated from Volvariella volvacea. Biochem J. 1997, 323 ( Pt 2) 557-565. Huang, Q.; Zhang, L.; Cheung, P.; Tan, X. Evaluation of sulfated α-glucans from Poria cocos mycelia as potential antitumor agent. Carbohyd Polym. 2006, 64 (2), 337-344. Huang, Y. C.; Chang, W. L.; Huang, S. F.; Lin, C. Y.; Lin, H. C.; Chang, T. C. Pachymic acid stimulates glucose uptake through enhanced GLUT4 expression and translocation. European Journal of Pharmacology. 2010, 648 (1-3), 39-49. Jin, H.; He, R.; Oyoshi, M.; Geha, R. S. Animal models of atopic dermatitis. J Invest Dermatol. 2009, 129 (1), 31-40. 41 Kang, H.; Lee, S.; Shin, D.; Lee, M.; Han, D.; Baek, N.; Son, K.; Kwon, B. Dehydrotrametenolic acid selectively inhibits the growth of H-ras transformed rat2 cells and induces apoptosis through caspase-3 pathway. Life Sci. 2006, 78 (6), 607-613. Ke, R. D.; Lin, S. F.; Chen, Y.; Ji, C. R.; Shu, Q. G. Analysis of chemical composition of polysaccharides from Poria cocos Wolf and its anti-tumor activity by NMR spectroscopy. Carbohyd Polym. 2010, 80 (1), 31-34. Kim, G. D.; Kim, T. H.; Jang, A. H.; Ahn, H. J.; Park, Y. S.; Park, C. S. alpha-Lipoic acid suppresses the development of DNFB-induced atopic dermatitis-like symptoms in NC/Nga mice. Exp Dermatol. 2011, 20 (2), 97-101. Lee, K. Y.; Jeon, Y. J. Polysaccharide isolated from Poria cocos sclerotium induces NF-kappaB/Rel activation and iNOS expression in murine macrophages. Int Immunopharmacol. 2003, 3 (10-11), 1353-1362. Li, T. H.; Hou, C. C.; Chang, C. L.; Yang, W. C. Anti-Hyperglycemic Properties of Crude Extract and Triterpenes from Poria cocos. Evid Based Complement Alternat Med. 2011, 2011 Lindequist, U.; Niedermeyer, T. H.; Julich, W. D. The pharmacological potential of mushrooms. Evid Based Complement Alternat Med. 2005, 2 (3), 285-299. Lu, M.-K.; Cheng, J.-J.; Lin, C.-Y.; Chang, C.-C. Purification, structural elucidation, and anti-inflammatory effect of a water-soluble 1,6-branched 1,3-α-d-galactan from cultured mycelia of Poria cocos. Food Chem. 2010, 118 (2), 349-356. Matsuda, H.; Watanabe, N.; Geba, G. P.; Sperl, J.; Tsudzuki, M.; Hiroi, J.; Matsumoto, M.; Ushio, H.; Saito, S.; Askenase, P. W.; Ra, C. Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice. Int Immunol. 1997, 9 (3), 461-466. 42 Narui, T.; Takahashi, K.; Kobayashi, M.; Shibata, S. A polysaccharide produced by laboratory cultivation of Poria cocos Wolf. Carbohydr Res. 1980, 87 (1), 161-163. Ooi, V. E.; Liu, F. Immunomodulation and anti-cancer activity of polysaccharide-protein complexes. Curr Med Chem. 2000, 7 (7), 715-729. Sato, M.; Tai, T.; Nunoura, Y.; Yajima, Y.; Kawashima, S.; Tanaka, K. Dehydrotrametenolic acid induces preadipocyte differentiation and sensitizes animal models of noninsulin-dependent diabetes mellitus to insulin. Biol Pharm Bull. 2002, 25 (1), 81-86. Shibata, S.; Yamazaki, M. The biogenesis of plant products. I. The biogenesis of rutin. Chem Pharm Bull (Tokyo). 1958, 6 (1), 42-45. Spergel, J. M.; Mizoguchi, E.; Brewer, | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32262 | - |
dc.description.abstract | 茯苓 (Poria cocos) 免疫調節蛋白 PCP,是分子量為 35.6 kDa 的醣蛋白,已知可藉由 TLR4-MyD88 途徑活化小鼠腹腔巨噬細胞,另外在有 anti CD3/CD28 存在下,PCP可活化 CD4+ 與 CD8+ 細胞的增生,故 PCP 為一具有免疫調節活性潛力的物質。為了進一步了解 PCP 的免疫調節機制與生化活性,故本研究分為兩個部分,第一部分欲選殖 PCP 的基因序列以幫助了解 PCP 的生化特性或其活性位置;第二部分則是利用類異位性皮膚炎小鼠動物模式,瞭解餵食 PCP 是否會減緩過敏小鼠的症狀。在第一部分的結果,根據 PCP 的 N 端胺基酸序列合成 PCP 之 degenerate 引子,再以 3’ 及 5’ Rapid amplification cDNA end (RACE) 的方法,選殖出全長為 807 bp 的 PCP cDNA,此序列的 ORF 為 579 bp,轉譯後可推衍出 194 胺基酸,預測分子量為 12.765 kDa。將此序列轉形至大腸桿菌株表現出 PCP 重組蛋白 (His)6-PCP,再利用 anti-His 與 anti-PCP 抗體進行西方轉漬法分析,兩種抗體皆可在 17 kDa 到 21 kDa 之間辨認到條帶。在第二部分試驗結果,餵食 PCP 組能降低淋巴結細胞分泌 IL-4 與 IL-5 的分泌量,但 IFN-γ 含量因故無法判定;此外餵食 PCP 會降低致敏鼠血清中的 OVA-specific IgG1 含量,同時增加血清中 OVA-specific IgG2a 的含量。在血清中 OVA-specific IgE 含量檢測,與正控制組相比,餵食 PCP 組能降低其含量。故推測餵食 PCP 處理可降低 OVA 致敏所引起的體內 Th2 免疫反應。綜合以上結果,本研究選殖出 PCP 的基因序列,與初步推測在類異位性皮膚炎動物模式中,餵食 PCP 可抑制致敏鼠體內免疫平衡趨向 Th2 免疫反應,改善過敏情況。 | zh_TW |
dc.description.abstract | Poria cocos immunomodulatory protein, PCP, was a glycoprotein with a molecular weight of 35.6 kDa. In murine models, PCP not only could activate primary macrophages through TLR4-MyD88 pathway, but also could enhance the proliferation of CD4+ and CD8+ cells in the presence of both CD3/CD28 antibodies. These studies showed that PCP was a potential immunomodulatory protein. The objectives of this study were first, to clone the cDNA of PCP, and second, to use an atopic dermatitis-like (AD-like) model to investigate whether administration of PCP could down-regulated the disease symptoms of mouse. In the first part, we designed the PCP degenerate primers based on their N-terminal amino acid sequence. Using the 3’ and 5’ Rapid amplification cDNA end (RACE) method, the cDNA sequence of PCP was cloned. The full length of PCP cDNA was 807 bp, and the ORF contained 579 bp nucleotide acids, encoding 194 amino acids. Predicted molecular weight of ORF encoding amino acid was 12.765 kDa. The ORF sequence obtained from PCP cDNA was further transformed into an E. coli expression system to produce PCP recombinant protein, (His)6-PCP. In the Western blotting analysis with anti-His and anti-PCP monoclonal antibodies, both antibodies recognized (His)6-PCP between the molecular weight 17 kDa to 26 kDa, hence to confirm the accuracy of PCP. In second part, oral administration of PCP suppressed the levels of IL-4 and IL-5 in draining LNs. However, the expression of IFN-γ was failed to be detected in the draining LNs. Oral administration of PCP suppressed the level of serum OVA-specific IgG1 but up-regulated the level of IgG2a. In the case of OVA specific IgE, oral administration of PCP could suppress IgE secretion in comparison with the positive control. These results suggested that the oral administration of PCP could suppress the OVA-induced Th2 response. Taken together, we this study cloned the cDNA of PCP and suggested that PCP could inhibit OVA-induced Th2 response to suppress a symptom of atopic dermatitis. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T03:39:32Z (GMT). No. of bitstreams: 1 ntu-100-R98628212-1.pdf: 6853342 bytes, checksum: 4d8b9cfcf152d0c7f62f0f11dbed376a (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 目錄
中文摘要 ....................................................................................................................................... I Abstract ........................................................................................................................................ II 目錄 ............................................................................................................................................ IV 圖目錄 ..................................................................................................................................... VIII 表目錄 ......................................................................................................................................... X 第一章 研究背景 .................................................................................................................. 1 第一節 概論 .......................................................................................................................... 1 第二節 茯苓之生理活性 ...................................................................................................... 2 一、 茯苓之簡介 .............................................................................................................. 2 二、 茯苓之成分組成 ...................................................................................................... 3 三、 茯苓中已知機能性成分 .......................................................................................... 3 (一) 茯苓多醣 ................................ ................................ ............................... 3 (二) 茯苓酸 (pachymic acid)................................ ................................ ........ 4 (三) 茯苓免疫調節蛋白 (P. cocos immunomodulatory protein, PCP) ....... 5 四、 茯苓之藥理作用 ...................................................................................................... 5 (一) 止吐 ................................ ................................ ................................ ....... 6 (二) 抑制癌細胞及抗腫瘤作用 ................................ ................................ ... 6 (三) 抑制發炎 ................................ ................................ ............................... 6 (四) 降血糖 ................................ ................................ ................................ ... 7 (五) 抑制腎臟炎 ................................ ................................ ........................... 7 第三節 Rapid amplification of cDNA ends, RACE 原理 .................................................... 7 第四節 異位性皮膚炎 .......................................................................................................... 8 V 一、過敏簡介 ................................................................................................................... 8 二、過敏反應類型 ........................................................................................................... 8 (一) 第一型過敏反應 ................................ ................................ ................... 8 (二) 第二型過敏反應 ................................ ................................ ................... 8 (三) 第三型過敏反應 ................................ ................................ ................... 9 (四) 第四型過敏反應 ................................ ................................ ................... 9 三、異位性皮膚炎 (atopic dermatitis, AD) .................................................................... 9 四、動物模式 ................................................................................................................... 9 第五節 研究動機與架構 .................................................................................................... 10 第二章 材料與方法 ................................................................................................................... 11 第一節 茯苓免疫調節蛋白之萃取 .................................................................................... 11 一、 蛋白萃取 ................................................................................................................ 11 二、 離子交換樹脂層析 ................................................................................................ 12 三、 快速蛋白質層析系統分析 .................................................................................... 12 四、 透析 ........................................................................................................................ 12 第二節 茯苓蛋白之生化特性 ............................................................................................ 12 一、 SDS-膠體電泳法 (SDS-polyacrylamide slab gel electrophoresis) ....................... 13 (1) 13.5 % polyacrylamide gel 製備 ................................ ........................ 13 (2) 電泳操作與膠片染色及褪染................................ ............................. 14 二、 醣蛋白染色分析 (periodic acid-Schiff stain) ....................................................... 14 三、 西方轉漬分析 ........................................................................................................ 14 第三節 免疫調節活性試驗 ................................................................................................ 15 VI 一、 茯苓蛋白對巨噬細胞之影響 ................................................................................ 15 (1) 巨噬細胞取得與培養 ................................ ................................ ......... 15 (2) ELISA 檢測細胞激素之產量 檢測細胞激素之產量 ................................ ............................ 16 二、 茯苓蛋白對淋巴細胞之影響 ................................................................................ 16 (1) 細胞激素 IFN-γ 測定 .................................................................................. 16 (2) 細胞增生試驗 ............................................................................................... 16 第四節 茯苓免疫調節蛋白基因之選殖 ............................................................................ 17 一、 RACE ready cDNA 之合成 .................................................................................. 18 (1) RNA 抽取 ................................ ................................ ........................... 18 (2) 3’ RACE ready cDNA 3’合成 ................................ ................................ 18 (3) 5’ RACE ready cDNA 5’合成 ................................ ................................ 19 二、 PCP 專一性引子之設計 ....................................................................................... 19 三、 RACE PCR ............................................................................................................. 19 四、 切膠純化 ................................................................................................................ 20 五、 T-A cloning ............................................................................................................. 20 六、 大腸桿菌之轉形與藍白篩選 ................................................................................ 20 七、 序列分析 ................................................................................................................ 21 第五節 重組蛋白基因之構築與表現 ................................................................................ 21 一、 cDNA 之合成 ........................................................................................................ 22 (1) RNA 抽取 ................................ ................................ ........................... 22 (2) RT -PCRPCRPCR ................................ ................................ ................................ 22 二、 載體構築及轉形 .................................................................................................... 23 VII (1) 抽取 plasmid plasmid ................................ ................................ ....................... 23 (2) 限制酶切位反應與接合 ................................ ................................ ..... 23 (3) 載體之轉形 ................................ ................................ ......................... 23 三、 重組蛋白質之誘導表現 ........................................................................................ 23 四、 重組蛋白質之純化 ................................................................................................ 24 第六節 類異位性皮膚炎動物試驗 .................................................................................... 24 一、類異位性皮膚炎試驗設計 ..................................................................................... 25 二、餵食茯苓蛋白對過敏小鼠之影響 ......................................................................... 25 (1) 細胞激素測定 ................................ ................................ ..................... 26 (2) 血清中特異性 IgE 檢測 ................................ ................................ .... 26 (3) 血清中特異性 IgG subtype 檢測 ................................ ...................... 26 第三章 結果 ............................................................................................................................... 28 第一節 茯苓免疫調節蛋白之萃取與生化特性 ................................................................ 28 第二節 茯苓免疫調節蛋白免疫調節活性試驗 ................................................................ 28 第三節 茯苓免疫調節蛋白基因之選殖 ............................................................................ 29 第四節 重組蛋白基因之構築與表現 ................................................................................ 31 第五節 類異位性皮膚炎動物試驗 .................................................................................... 32 第四章 討論 ............................................................................................................................... 34 第五章 參考文獻 ....................................................................................................................... 38 VIII 圖目錄 Fig. 1. Biochemical characteristics of PCP. ................................................................................ 45 Fig. 2. Effect of PCP on cytokine released by peritoneal macrophage. ...................................... 46 Fig. 3. Effect of PCP on mouse splenocyte activation. ............................................................... 47 Fig. 4. 3’ RACE PCR product with primer 320. ......................................................................... 48 Fig. 5. Nucleotide sequence and translation result of primer 320 product. ................................. 49 Fig. 6. 5’ RACE PCR product of primer 5-320-2. ...................................................................... 50 Fig. 7. Nucleotide sequence and translation result of primer 5-320-2 product. .......................... 51 Fig. 8. Alignment of deduced amino acid sequence and N-terminal amino sequence of PCP. . 52 Fig. 9. Multiple sequence alignment of 3’ RACE PCR products.. .............................................. 53 Fig. 10. Multiple sequence alignment of 5’ RACE PCR products. ............................................. 54 Fig. 11. Full length cDNA. .......................................................................................................... 55 Fig. 12. Theoretical pI/Mw for amino acid sequence deduced from cDNA sequence terminated translation at different stop codon. .............................................................................................. 56 Fig. 13. Signal P predictions of PCP. .......................................................................................... 57 Fig. 14. YinOYang 1.2 prediction results of PCP. ...................................................................... 58 Fig. 15. NetNGlyc 1.0 prediction results of PCP. ....................................................................... 59 Fig. 16. The protein-protein blast analysis for PCP. ................................................................... 60 Fig. 17. The map of pET30 expression vector. ........................................................................... 61 Fig. 18. Expression of His-tag fusion protein PCP. .................................................................... 62 Fig. 19. Expression of His-tag fusion protein PCP. .................................................................... 63 IX Fig. 20. Effect of PCP on cytokine release from lymphonode in epicutaneous immune response. .................................................................................................................................................... 64 Fig. 21. Analysis of the effect on epicutaneous immune response with oral PCP. ..................... 65 X 表目錄 Table 1. PCP-specific primer used in 3’ RACE reaction. ........................................................... 66 Table 2. PCP-specific primer used in RACE PCR. ..................................................................... 66 Table 3. PCP-specific primer used in restriction enzyme digestion design. ............................... 66 Table 4. The result of protein-protein blasts analysis of PCP. .................................................... 67 | |
dc.language.iso | zh-TW | |
dc.title | 茯苓免疫調節蛋白之基因選殖及其對類異位性皮膚炎老鼠模式之效應 | zh_TW |
dc.title | Molecular Cloning of the Immunomodulatory Protein PCP and its Effect in Atopic Dermatitis-Like Mouse Model | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 周志輝,蔣恩沛,繆希椿 | |
dc.subject.keyword | 免疫調節蛋白,茯苓,RACE PCR,OVA,異位性皮膚炎, | zh_TW |
dc.subject.keyword | immunomodulatory protein,Poria cocos,RACE PCR,OVA,atopic dermatitis, | en |
dc.relation.page | 67 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-07-29 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 園藝學研究所 | zh_TW |
顯示於系所單位: | 園藝暨景觀學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-100-1.pdf 目前未授權公開取用 | 6.69 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。