請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90604完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭光成 | zh_TW |
| dc.contributor.advisor | Kuan-Chen Cheng | en |
| dc.contributor.author | 黃筱筑 | zh_TW |
| dc.contributor.author | Hsiao-Chu Huang | en |
| dc.date.accessioned | 2023-10-03T16:49:30Z | - |
| dc.date.available | 2023-11-09 | - |
| dc.date.copyright | 2023-10-03 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-08 | - |
| dc.identifier.citation | 郭耀綸 (2008)。紅藜推廣手冊。屏東科技大學。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90604 | - |
| dc.description.abstract | 衰老是一種隨著時間的推移、年齡的增長所發生的自然現象，同時由於細胞和組織中各種有害變化的累積所引發的現象。當生物體暴露於某些外源性物質，可能導致內源性和外源性 ROS 之間不平衡，從而降低抗氧化防禦能力而引起衰老等現象發生。本研究目的為評估自發酵發芽臺灣藜中所分離出之 < 2 kDa 小分子胜肽產量於生物反應器發酵之最適發酵天數，並進一步鑑定其序列，運用 UVA 誘導細胞光衰老及動物模式生物秀麗隱桿線蟲評估胜肽延緩衰老之功效。將臺灣藜發芽四天並且接種 Rhizopus oligosporus 於生物反應器進行發酵，以發酵第四天為最適生產胜肽之天數，產量增加 5.89 倍。依據快速蛋白質液相層析圖譜得知同樣於發酵第 4 天有最高之 UV 280 nm 吸光值933.27，相比第 0 天增加 3.08 倍；透過蛋白質鑑定結果得知此胜肽之序列為 GGGGGKP (GRP)。評估 GRP 經 UVA 誘導 Hs68 細胞光衰老之保護效果，50 及 100 ug/mL GRP 分別回復細胞存活率至 85.52±14.96% 及 83.45±7.83%，增加 1.29 及 1.26 倍；並且分別減少 ROS 相對螢光強度 1.85 及 2.46 倍；於衰老指標酵素活性 SA-β‐gal 分別減少 8.13 及 7.00 倍。利用 RNA-seq 之基因富集結果推測預先添加 GRP 可能具有減緩經 UVA 所誘導之氧化壓力，因而回復細胞週期停滯，接著 mRNA 表現量顯示，50 及 100 ug/mL GRP 分別顯著增加 Nrf2 基因 3.96 與 10.69 倍的表現，此外其下游 HO-1 基因亦分別提升 6.67 與 9.30 倍，表示其可能透過 Nrf2 途徑減少 ROS 生成；另外於 50 及 100 ug/mL兩種 GRP 濃度可顯著降低 p53 基因表現達 4.09 及 2.69 倍 並且可延緩 p21 基因表現至 14.15 與 24.00 倍；接著利用 ChIP 分析，相比控制組，分別顯著上調各基因 GSR 61.94 與 28.79 倍、SOD 8.37 與 16.76 倍 及 NQO1 3.63 與 3.98 倍，證實 GRP 透過 Nrf2 轉錄因子調節途徑。研究顯示 GRP 有助於緩解 UVA 誘導之細胞週期停滯，達到延緩細胞衰老之功效。50 及 100 ug/mL GRP 相較於控制組，顯著延長秀麗隱桿線蟲之平均生命週期分別顯著提升 13.4% 及 11.0%，使生存曲線向右移；同時顯著增加咽部抽動速率 1.08 與 1.35 倍，以及改善 1.33 與 1.34 倍由促氧化劑誘導氧化壓力的生存率，接著 mRNA 表現量相較於控制組，50 ug/mL GRP 分別顯著提升 skn-1 下游基因 gcs-1、gst-4 與 gst-7 ( 3.3 倍、2.32 倍和 6.00 倍 )。本研究證實發酵發芽臺灣藜胜肽於體內及體外試驗皆具有延緩衰老之功效。 | zh_TW |
| dc.description.abstract | Population aging is an international concern. Taiwan has confronted an aging society since 1993. By the end of January 2021, the proportion of 65-year-olds has accounted for 16.2%, over 14% of the aging society defined by the United Nations. The purpose of this study is to evaluate the optimal fermentation days of < 2 kDa peptides isolated from fermented Chenopodium formosanum sprouts. Then evaluate the anti-aging effect of peptides through UVA-irradiation and the animal model Caenorhabditis elegans. The fourth day of fermentation was the optimum day for peptide production, and the yield increased by 5.89 times. The sequence is GGGGGKP (GRP), which is identified by protein identificaiton. To evaluate the protective effect of GRP on UVA-induced aging of Hs68 cells, 50 and 100 ug/mL GRP restored cell viability to 85.52±14.96% and 83.45±7.83%, respectively, increasing by 1.29 and 1.26 times; and reduced the ROS fluorescence intensity by 1.85 and 2.46 times; the aging indicator enzyme activity SA-β‐gal is reduced by 8.13 and 7.00 times respectively. Using the Gene Ontology results of RNA-seq, it is speculated that the pretreatment with GRP may relieve the oxidative stress induced by UVA irradiation, thereby restoring cell cycle arrest. Then mRNA expression showed that 50 and 100 ug/mL GRP significantly increased the overall gene expression of Nrf2 by 3.96, 10.69 times and its downstream HO-1 gene by 6.67, 9.30 times, respectively, indicating that the ROS generation may be reduced through the Nrf2 pathway. The gene expression of p53 was increased by 4.09, 2.69 times, p21 was improved by 14.15, 24.00 times, help to relieve UVA-induced cell cycle arrest and achieve the effect of delaying cell aging. Then, using ChIP analysis, compared with the control group, the genes GSR was significantly up-regulated by 61.94 and 28.79 times, SOD by 8.37 and 16.76 times, and NQO1 by 3.63 and 3.98 times, confirming that GRP regulates the pathway through the Nrf2 transcription factor. Compared with the control group, 50, 100 ug/mL GRP significantly prolong the average lifespan of C. elegans by 13.4±3.78 %, 11.0±2.77 % and increase the pharyngeal pumping rate by 1.08, 1.35 times. At the same time, it could improve survival of oxidative stress induced by juglone by 1.33, 1.34 times. Then, 50 ug/mL GRP increases gene expression of gcs-1, gst-4, gst-7 by 3.3, 2.32 and 6.00 times, respectively. This study confirmed that the fermented and germinated Chenopodium formosanum peptide has the effect of delaying aging both in vivo and in vitro. | en |
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| dc.description.provenance | Made available in DSpace on 2023-10-03T16:49:30Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 謝誌 I
摘要 II Abstract IV 目錄 VI 圖目錄 XII 表目錄 XIV List of Figures XV List of Tables XVII 壹、 前言 1 貳、 文獻回顧 2 2.1 老化 2 2.1.1 衰老簡介及與慢性疾病關聯 2 2.1.2 我國高齡化現況 2 2.1.3 細胞衰老成因 2 2.1.4 細胞衰老可能機制 3 2.2 氧化壓力 6 2.2.1 簡介 6 2.2.2 活性含氧物的產生 6 2.2.3 活性含氧物對於生物體的影響 6 2.2.4 延緩衰老可能機制 7 2.3 臺灣藜 8 2.3.1 藜麥簡介 8 2.3.2 臺灣藜簡介 9 2.3.3 一般營養成分 9 2.3.4 機能性成分 10 2.3.5 具延緩衰老之成分 12 2.3.6 種子發芽的優點 13 2.4 發酵 13 2.4.1 定義 13 2.4.2 液態發酵 14 2.4.3 固態發酵 14 2.4.4 少孢根黴菌 (Rhizopus oligosporus) 15 2.4.5 Rhizopus oligosporus 發酵藜麥 16 2.5 生物活性胜肽 17 2.5.1 臺灣藜蛋白質 17 2.5.2 製備活性胜肽 18 2.5.3 抗氧化活性胜肽與延緩老化研究 20 2.6 誘導細胞光衰老 21 2.6.1 人類纖維母細胞 21 2.6.2 紫外線 (UV) 21 2.6.3 誘導細胞衰老 22 2.6.4 細胞光衰老機制 23 2.7 秀麗隱桿線蟲 (C. elegans) 23 2.7.1 簡介 23 2.7.2 衰老之生物指標 (Biomarker of aging) 24 2.7.3 調節線蟲壽命因素之研究 25 2.8 次世代定序 (Next Generation Sequencing, NGS) 27 2.8.1 次世代定序技術 27 2.8.2 高通量核糖核酸定序 (RNA-sequencing) 27 2.9 具延緩衰老功效之健康食品 28 2.9.1 健康食品之延緩衰老保健評估方法 28 2.9.2 相關之高齡食品及開發 28 參、 研究目的與實驗架構 29 3.1 研究目的 29 3.2 實驗架構 30 肆、 實驗材料研究目的與實驗架構 32 4.1 實驗材料 32 4.1.1 實驗原料 32 4.1.2 實驗菌株 32 4.1.3 實驗細胞株 32 4.1.4 細胞實驗材料 32 4.1.5 培養基配置藥品 33 4.1.6 分析實驗藥品 33 4.1.7 培養基配置 34 4.1.8 儀器設備 36 4.2 實驗方法 37 4.2.1 原料臺灣藜發芽 37 4.2.1.1 培養基配置 37 4.2.1.2 R. oligosporus 菌株活化 37 4.2.1.3 種菌培養 37 4.2.1.4 孢子懸浮液製作 37 4.2.2 固態發酵產物製備 37 4.2.2.1 以生物反應器培養 R. oligosporus 發酵發芽臺灣藜 37 4.2.3 分離純化發芽臺灣藜之發酵產物中的蛋白質及胜肽 38 4.2.3.1 發酵產物脫脂 38 4.2.3.2 粗蛋白質萃取 38 4.2.3.3 硫酸胺鹽析 38 4.2.3.4 以 FPLC 脫鹽分離純化 < 2 kDa 胜肽 40 4.2.3.5 蛋白質及胜肽之產率計算 42 4.2.4 蛋白質身分鑑定 (Protein identification) 42 4.2.4.1 小於 2 kDa 胜肽樣品前處理 42 4.2.4.2 胜肽身分鑑定 44 4.2.5 紫外線誘導光衰老對 Hs68 細胞試驗 46 4.2.5.1 實驗細胞株及細胞培養基 46 4.2.5.2 細胞計數 46 4.2.5.3 Hs68 細胞凍管保存 46 4.2.5.4 細胞實驗樣品前處理 47 4.2.5.5 細胞存活率之測定 48 4.2.5.6 建立UVA照射致 Hs68 細胞光老化平台 48 4.2.5.7 細胞內 ROS 測定 48 4.2.5.8 細胞SA-β-gal之測定 49 4.2.5.9 染色質免疫沉澱Chromatin immunoprecipitation (ChIP) 49 4.2.5.9.1 Hs68 細胞樣品前處理 49 4.2.5.9.2 染色質免疫沉澱 49 4.2.6 RNA 定序資料分析 51 4.2.6.1 Hs68 細胞樣品製備 51 4.2.6.2 萃取 Hs68 細胞 RNA 51 4.2.7 高通量核糖核酸定序 (RNA-Sequencing) 52 4.2.8 建立以 C. elegans 為模式動物之延緩衰老平台 53 4.2.8.1 品系來源、保存與解凍 53 4.2.8.2 C. elegans 保存與解凍 53 4.2.8.3 E. coli OP50 活化及含有 E. coli OP50 之 NGM agar 之製備 53 4.2.8.4 蟲卵分離及同步化 54 4.2.8.5 利用胜肽以培養 C. elegans 之相關試驗 55 4.2.8.5.1 樣品前處理 55 4.2.8.5.2 延長壽命測試 (Lifespan assay) 55 4.2.8.5.4 咽部抽動速率計數 (Pharyngeal pumping rate) 56 4.2.8.5.5 抵禦壓力能力 56 4.2.9 相關基因表現測定 57 4.2.9.1 萃取 RNA 57 4.2.9.2 RNA 反轉錄成 cDNA 57 4.2.9.3 即時定量聚合酶連鎖反應 (Quantitative Real-Time PCR, qPCR) 58 4.2.10 統計分析 60 伍、 結果與討論 61 5.1 發芽臺灣藜胜肽於生物反應器 (Bioreactor) 發酵最適條件 61 5.1.1 發芽臺灣藜之條件參考 61 5.1.2 R. oligosporus 接種條件參考 61 5.1.3 於生物反應器發酵發芽臺灣藜之外觀型態 61 5.1.4 臺灣藜 < 2 kDa胜肽之產率及快速蛋白質液相層析圖譜 64 5.2 小於 2kDa 胜肽之分子量區間證明及序列鑑定結果 67 5.3 評估胜肽經 UVA 誘導 Hs68 光衰老之保護效果 69 5.3.1 臺灣藜胜肽對 Hs68 細胞毒性 69 5.3.2 GRP 對 UVA 誘導 Hs68 細胞存活率及 ROS 產生之影響 70 5.3.3 利用 RNA-Sequencing 分析 GRP 減緩 UVA 誘導細胞衰老轉錄體分析 73 5.3.3.1 基因表達差異 (Differential Expressing Genes, DEGs) 73 5.3.3.2 基因富集功能差異分析 (Gene Ontology, GO) 75 5.3.4 GRP 對 UVA 誘導 Hs68 細胞之 SA--gal 之影響 81 5.3.5 GRP 對調節 UVA 誘導細胞衰老的基因表現 83 5.3.6 GRP 對調節 UVA 誘導細胞衰老的 ChIP 分析 86 5.4 評估 GRP 延緩秀麗隱桿線蟲模式生物衰老之功效 89 5.4.1 生命週期 (Lifespan assay) 89 5.4.2 咽部抽動速率計數 (Pharyngeal pumping rate) 91 5.4.3 壓力抵抗能力試驗 (Resistance to oxidative stress) 93 5.4.4 GRP 延長線蟲壽命之基因表現 95 陸、 結論與未來展望 97 柒、 參考文獻 99 捌、 附錄 123 附錄一、論文原創性比對 123 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 延緩衰老 | zh_TW |
| dc.subject | 臺灣藜 | zh_TW |
| dc.subject | 秀麗隱桿線蟲 | zh_TW |
| dc.subject | 固態發酵 | zh_TW |
| dc.subject | 胜肽 | zh_TW |
| dc.subject | Caenorhabditis elegans | en |
| dc.subject | Chenopodium formosanum | en |
| dc.subject | solid-state fermentation | en |
| dc.subject | anti-senescence | en |
| dc.subject | peptide | en |
| dc.title | 評估固態發酵發芽臺灣藜胜肽其延緩衰老之功效 | zh_TW |
| dc.title | Evaluation of Solid-state Fermentation of Chenopodium formosanum Sprouts Peptide with Anti-senescence Activity | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 郭星君;陳勁初;游舒涵;黃崇雄 | zh_TW |
| dc.contributor.oralexamcommittee | Hsing-Chun Kuo;Chin-Chu Chen;Shu-Han Yu;Chung-Hsiung Huang | en |
| dc.subject.keyword | 延緩衰老,秀麗隱桿線蟲,臺灣藜,固態發酵,胜肽, | zh_TW |
| dc.subject.keyword | anti-senescence,Caenorhabditis elegans,Chenopodium formosanum,solid-state fermentation,peptide, | en |
| dc.relation.page | 150 | - |
| dc.identifier.doi | 10.6342/NTU202302822 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2023-08-09 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 食品科技研究所 | - |
| 顯示於系所單位： | 食品科技研究所 | |
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