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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃青真 | |
| dc.contributor.author | Wen-Chia Feng | en |
| dc.contributor.author | 馮文嘉 | zh_TW |
| dc.date.accessioned | 2021-06-16T23:42:07Z | - |
| dc.date.available | 2018-07-24 | |
| dc.date.copyright | 2012-08-01 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-07-24 | |
| dc.identifier.citation | Abdollahi, M., Zuki, A.B., Goh, Y.M., Rezaeizadeh, A., and Noordin, M.M. (2011). Effects of Momordica charantia on pancreatic histopathological changes associated with streptozotocin-induced diabetes in neonatal rats. Histology and histopathology 26, 13-21.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65425 | - |
| dc.description.abstract | 第二型糖尿病病發病前會經歷一段正常與糖尿病間的過渡時期，稱之為糖尿病前期。目前研究認為，糖尿病前期為『飲食介入』預防糖尿病發生之重要時期。過去本研究室發現，苦瓜經 β-glucosidase 處理後的水萃物和小分子乙酸乙酯萃物比未經酵素處理的樣品更具有降血糖活性，但其詳細降血糖有效成分與機制則尚未被確認。因此，本研究建立以螢光標定葡萄糖，2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4yl) amino]-2-deoxyglucose ( 2-NBDG )，作為偵測肝細胞株 FL83B 汲取葡萄糖模式平台；並以胰臟 beta 細胞之胰島素分泌作為模式平台，來測試苦瓜各式萃物、水解物、區分物及分離物之活性，並探討可能的活性成分與其影響細胞的可能機制。
結果顯示，FL83B 肝臟細胞以處理 15 小時之條件最能顯現苦瓜萃物促葡萄糖汲取之活性。水解苦瓜樣品中以強酸水解產物增加 FL83B 細胞汲取葡萄糖的量較酵素水解產物高。弱酸水解區分物皆顯著抑制細胞對葡萄糖之汲取。胰島beta細胞株部分，唯果汁正丁醇萃物與其經酵素水解的產物可顯著增加細胞分泌胰島素，其餘萃物或區分物沒有顯著影響或有顯著抑制效果。代謝體學分析比較經山苦瓜水萃物處理之細胞與對照組細胞，推測，可能受到樣品影響的代謝路徑，在 FL83B 中為glycerophospholipid、purine、glutathione 及 phenylalanine 的代謝；在 HIT-T15 細胞中則為 purine、glutathione、taurine and hypotaurine、cysteine 及 methionine 的代謝。另外，本研究亦發現adenyl cyclase 抑制劑 SQ22536 可部分抑制HIT-T15 細胞受山苦瓜果汁萃物刺激之胰島素分泌，而 ATP 敏感性鉀通道開放劑 diazoxide、phosphollipase C 抑制劑 U73122 以及 proteinkinase A 抑制劑H89則否。由本研究的結果可知山苦瓜萃物具調節血糖潛力的有效成分似非單一化合物，而其的確可有效的調節肝臟細胞對葡萄糖的汲取並可部分透過影響 adenylyl cyclase 來促進胰臟 beta 細胞分泌胰島素。 | zh_TW |
| dc.description.abstract | Type 2 diabetes has become a significant public health concern. The “prediabetic” period precede the development of type 2 diabetes are critical for dietary interventions. The water extract(WE) of Bitter gourd (Momordica charantia,MC) (BG) and the ethyl acetate (EA) extract of hydrolyzed small molecules fraction of WE were shown to enhance GLP-1 secretion in STC-1, an enteroendocrine cell line, increase insulin secretion in beta-cell lines, and promote glucose uptake in hepatocyte, adipocytes and myocytes. But specific hypoglycemic active ingredients and mechanisms have not been confirmed. This study thus aimed to examining in more details the activities of hydrolyzed extracts/fractions/isolates of wild BG (WBG) using glucose uptake of hepatocytes ( FL83B hepatic cell line) and insulin secretion of islet beta cells (HIT-T15 beta cell line). Possible mechanisms were preliminarily explored by using metabolomics analysis and specific inhibitor in insulin secretion pathway.
The glucose uptake of FL83B cells was determined by using the uptake of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG), a fluorescent analog of d-glucose. Pre-treatment of FL83B cells with WBG water extracts for 15 hr was found to significantly increased glucose uptake. Among various hydrolyzed extracts/fractions/isolates of WBG, the hexane extract of the 15% HCl hydrolyzeds product, butanol extract of enzyme hydrolyzed product and ethyl acetate extract of 2% HCl hydrolyzed product significantly enhanced glucose uptake of FL83B.In contrast, extracts of remaining hydrolyzed products, and fractions isolated significantly inhibited glucose uptake of FL83B. Insulin secretion was measured after HIT-T15 cells were treated with 10mM glucose or WBG extract/fractions for 1 hr. Compared to the results of glucose uptake of FL83B cells, insulin secretion was significantly increased only in cells treated with butanol extract of WBG juice, and the EA or butanol extracts of enzymatic hydrolysis products of WBG juice, suggesting different active compounds in WBG acting on different target cells. . LC-TOF-MS was used to compare metabolome of cells treated with WE, insulin or vehicles. Human metabolome dataset (HMDB) and KEGG database was used for predicting the regulated compounds and mapping possible biochemical pathways involved. The metabolic pathways affected include glycerophospholipid, purine, glutathione and phenylalanine metabolism in FL83B cell and purine, glutathione, taurine and hypotaurine, cysteine and methionine metabolism in HIT-T15 cell. Finally, the WBG juice stimulated insulin secretion of HIT-T15 cells was partially inhibited by the adenyl cyclase inhibitor SQ22536, but not by ATP-sensitive K channel agonist diazoxide, or phospholipase C inhibitor U73122 or protein kinase A inhibitor H89. The result suggested that WBG juice may stimulate insulin secretion in beta cells through activating adenylyl cyclase, at least in part. In conclusion, this study demonstrated that the hypoglycemic activity of WBG might not be attributed to a single compound and indeed, WBG extracts could enhance glucose uptake in hepatic cell line and increased insulin secretion of islet beta cell line through activating adenylyl cyclase, at least in part. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T23:42:07Z (GMT). No. of bitstreams: 1 ntu-101-R99b22012-1.pdf: 2794936 bytes, checksum: c945cbf89dd026d96d02e2a72d0d1018 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 中文摘要 i
Abstract ii 總目錄 iv 表目錄 ix 圖目錄 x 第一章 緒論 1 第一節 前言 1 第二節 文獻回顧 1 一、 第二型糖尿病 1 二、 血糖調控機制 4 (一) 胰島素的生成、分泌與作用 5 (二) 胰島素分泌障礙 7 (三) 胰島素阻抗 9 三、 肝臟血糖調控機制與胰島素抗性 12 四、 代謝體學 (metabolomics) 14 五、 苦瓜 17 第三節 實驗假說與設計 26 一、 FL83B 小鼠肝細胞株 26 (一) 葡萄糖汲取模式建立 26 (二) 樣品效用評估 26 二、 以 HIT-T15 倉鼠胰島 beta 細胞株評估樣品對胰島素分泌之影響 26 三、 山苦瓜萃物作用於肝細胞與胰島 beta 細胞之機制探討 26 (一) 代謝體學分析 26 (二) 樣品增加細胞分泌胰島素之機制探討 26 第二章 山苦瓜萃取物對小鼠肝細胞株 FL83B 汲取葡萄糖能力之影響 27 第一節 前言 27 第二節 材料與方法 28 一、 細胞株 28 二、 試劑與器材 28 (一) 試劑 28 (二) 器材 29 三、 山苦瓜萃取物暨其區分物製備 29 (一) 山苦瓜果汁正丁醇萃物製備 (H2JB / H3JB) 29 (二) 山苦瓜果汁正丁醇萃物區分樣品 29 四、 實驗方法 31 (一) 細胞繼代培養 31 (二) 葡萄糖汲取模式建立與最佳化 32 (三) 以不同苦瓜萃取物及其區分物處理細胞 33 (四) 螢光葡萄糖測定 34 (五) 蛋白質測定 34 (六) 細胞數測定 (MTT 染色) 34 第三節 實驗結果 35 一、 弱酸水解物 (3a2-E) 經 HPLC 逆相管柱區分後之結果 35 二、 葡萄糖汲取模式建立 (圖 2-4 與圖 2-5) 36 三、 山苦瓜各種萃取物及區分物對 FL83B 肝細胞株汲取葡萄糖能力之影響 37 (一) 山苦瓜果汁正丁醇萃物 (H2JB, H3JB) 與其酵素水解區分物 (2Be-E, 2Be-B, 3Be-E, 3Be-B) 37 (二) 山苦瓜果汁正丁醇萃物經強酸 (2Ba15-E, 2Ba15-H) 及弱酸水解產物 (3a2-E) 與其 HPLC 區分物 (RP1-6) 37 (三) 山苦瓜果汁正丁醇萃物經不同濃度酸水解與添加 Vit C 處理之樣品 (3a2c-E / 3a2-E / 3a15c-E / 3a15-E / 3a18c-E / 3a18-E / a15c-E) 37 第四節 討論 43 一、 模式建立與適應性 43 二、 山苦瓜萃取物對肝細胞汲取葡萄糖之影響 43 三、 山苦瓜萃取物增加肝細胞株 FL83B 汲取葡萄糖之可能機制探討 45 (一) 促使細胞汲取葡萄糖 45 (二) 促進細胞內葡萄糖的代謝 46 (三) 抑制糖質新生 46 第五節 結論 47 第三章 山苦瓜萃取物對 HIT-T15 倉鼠胰島 beta 細胞株分泌胰島素能力之影響 48 第一節 前言 48 第二節 材料與方法 48 一、 細胞株 48 二、 試劑與器材 49 (一) 試劑 49 (二) 器材 50 三、 山苦瓜萃取物暨其區分物製備 50 四、 實驗方法 50 (一) 細胞培養 50 (二) 葡萄糖促使 HIT-T15 細胞株分泌胰島素試驗 51 (三) 胰島素含量分析 (Mercodia AB, SE-754 50 Uppsala, Sweden) 51 (四) 蛋白質測定 52 五、 統計分析 52 第三節 結果 52 一、 山苦瓜果汁正丁醇萃物 (H2JB, H3JB) 與其酵素水解區分物 (2Be-E, 2Be-B, 3Be-E, 3Be-B) 52 二、 山苦瓜果汁正丁醇萃物經強酸 (2Ba15 -E, 2Ba15 -H) 及弱酸水解產物 (3Ba2 -E) 與其 HPLC 區分物 (RP1-6) 52 三、 山苦瓜果汁正丁醇萃物經不同濃度酸水解與添加 Vit C 處理之樣品 53 四、 自花連四號山苦瓜分離出之三萜類化合物（cpd1-6）(由本實驗室 徐瑨博士提供)(Hsu et al., 2011) 53 第四節 討論 58 一、 山苦瓜萃取物影響胰島 beta 細胞分泌胰島素之活性成分 58 二、 山苦瓜萃取物增加 HIT-T15 細胞分泌胰島素之可能機制探討 59 第五節 結論 59 第四章 山苦瓜萃物於肝臟細胞與胰島 beta 細胞之機制探 60 第一節 前言 60 第二節 材料與方法 60 一、 細胞株 60 二、 試劑與器材 61 (一) 試劑 61 (二) 器材 61 三、 山苦瓜萃取物製備 (由本實驗室 黃婷妮學姊製備) 62 (一) 代謝體學 (WE 與 Se) 62 (二) 樣品影響胰島素分泌機制探討 62 四、 實驗方法 62 (一) 細胞培養及樣品處理 62 (二) 細胞代謝物萃取 63 (三) LC-TOF-MS 及數據分析 63 第三節 結果 64 一、 代謝體學 64 二、 山苦瓜萃物影響胰島素分泌機制探討 69 第四節 討論 75 一、 代謝體學 75 二、 山苦瓜萃物影響胰島素分泌機制探討 77 第五節 結論 78 第五章 綜合討論與總結論 79 第一節 綜合討論 79 第二節 總結論 80 第六章 參考文獻 82 表目錄 表 1-1造成 IRS1 磷酸化的蛋白激酶、原因以及對胰島素訊息之影響 10 表 1- 2 自苦瓜分離之葫蘆烷型三萜類化合物降血糖活性相關文獻整理 21 表 1- 3 苦瓜對肝臟細胞與胰島 beta 細胞相關文獻整理 24 表 2-1 山苦瓜各萃取物刺激與抑制 FL83B 細胞汲取葡萄糖之最大倍數與濃度總整理 42 表 3-1 山苦瓜各萃取物刺激與抑制 HIT-T15 細胞分泌胰島素之最大倍數總整理 57 表 4-1、以山苦瓜萃物處理FL83B 細胞的代謝產物在各處理間的改變預測與可能參與的代謝物 67 表 4-2、以山苦瓜萃物處理HIT-T15 細胞的代謝產物在各處理間的改變預測與可能參與的代謝物 68 表 5-1、山苦瓜萃物樣品對肝臟細胞 FL83B 葡萄糖汲取與胰島 beta 細胞 HIT-T15 胰島素分泌影響整理表 81 圖目錄 圖 1-1 周邊組織中，賀爾蒙、營養素及代謝狀態對 AMPK 活性的影響 - 3 - 圖 1- 2 AMPK 對肝臟代謝的影響 4 圖 1- 3 胰島素轉譯修飾與分泌機制 6 圖 1-4 胰島素在細胞中所引起的訊息傳導以及作用 7 圖 1-5大鼠胰島素基因之啟動子上可被葡萄糖調節區域及相關轉錄因子 8 圖 1-6長期處於高糖及高脂之下對於胰島素基因表現的影響 8 圖 1-7胰島素分泌障礙與肥胖、胰島素抗性與第二型糖尿病之關係 9 圖 1- 8 IRS1之磷酸化對其功能的影響 11 圖 1-9肝臟中葡萄糖代謝的調控 13 圖 1-10胰島素阻抗對肝臟脂肪代謝的影響 14 圖 1-11在代謝體學研究中採用質譜儀與核磁共振儀分析步驟 16 圖 1-12 糖尿病鼠模式與第二型糖尿病罹病者體內受到影響的代謝路徑統整 16 圖 1-13山苦瓜對各種器官組織的影響 19 圖 1-14山苦瓜對碳水化合物與脂質代謝影響的可能機制 20 圖 2-1 山苦瓜區分流程 31 圖 2- 2 HPLC 逆相管柱區分弱酸水解樣品 3a2-E 35 圖 2- 3化合物 RP3 之結構 36 圖 2- 4 胰島素對 FL83B 肝細胞汲取 2-NBDG 之促進作用—2-NBDG 濃度、處理時間與處理方式之影響 38 圖 2- 5 FL83B 肝細胞以花蓮四號山苦瓜水萃物 (WE) 處理 (A) 30min (pretreat), 或 60min (co-treat) 或 (B) 15hr 後對 2-NBDG 之汲取 39 圖 2- 6 (A) 花蓮二號及 (B) 三號山苦瓜果汁正丁醇萃物與其酵素水解及酸水解萃物對 FL83B 細胞汲取葡萄糖之影響 40 圖 2-7 (A) 3a2-E 經 HPLC區分後所得 RP1-6 及 (B) 不同條件酸水解產物對 FL83B 細胞汲取葡萄糖之影響 41 圖 3-1 花蓮二號及三號山苦瓜正丁醇萃物與其酵素水解及酸水解萃物對 HIT-T15 細胞分泌胰島素之影響 54 圖 3- 2 (A) 3a2-E 經 HPLC區分後所得 RP1-6 及 (B) 不同條件酸水解產物對 HIT-T15 細胞分泌胰島素之影響 55 圖 3-3、自花蓮四號品系山苦瓜分離出之三萜類化合物（cpd1~6）對HIT-T15 細胞分泌胰島素之影響 56 圖 4-1、以 vehicle、insulin 或山苦瓜萃物 WE 及 Se 處理 FL83B 細胞株後其代謝物的主成分分析 65 圖 4-2、以 vehicle、insulin 或山苦瓜萃物 WE 及 Se 處理 HIT-T15 細胞株後其代謝物的主成分分析 66 圖 4-3、ATP 敏感性鉀通道開放劑 diazoxide (DZ) 對 HIT-T15 細胞分泌胰島素影響 70 圖 4-4、phospholipase c 抑制劑 U73122 對 HIT-T15 細胞分泌胰島素影響 71 圖 4-5、adenylyl cyclase 抑制劑 SQ22536 對 HIT-T15 細胞分泌胰島素影響 72 圖4-6、Phosphodiesterases 抑制劑 IBMX對 HIT-T15 細胞分泌胰島素影響 73 圖4-7、PKA 抑制劑H89對 HIT-T15 細胞分泌胰島素影響 74 | |
| dc.language.iso | zh-TW | |
| dc.subject | 糖尿病 | zh_TW |
| dc.subject | 肝臟細胞株 | zh_TW |
| dc.subject | 胰臟 β 細胞株 | zh_TW |
| dc.subject | 山苦瓜 | zh_TW |
| dc.subject | 代謝體學 | zh_TW |
| dc.subject | Type 2 diabetes | en |
| dc.subject | hepatic cell line | en |
| dc.subject | islet beta cell line | en |
| dc.subject | wild bitter gourd (Momordica Charantia) | en |
| dc.subject | metabolomics | en |
| dc.title | 以 FL83B 肝臟細胞株汲取葡萄糖及 HIT-T15 胰臟 β 細胞株胰島素分泌為平台研究山苦瓜之血糖調節活性萃物 | zh_TW |
| dc.title | Studies on the Extracts of Momordica charantia L. that enhance Glucose Uptake in FL83B Hepatic Cell Line and Insulin Secretion of HIT-T15 Islet Beta Cell Line | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林璧鳳,楊健志,李昆達,蕭明熙 | |
| dc.subject.keyword | 糖尿病,肝臟細胞株,胰臟 β 細胞株,山苦瓜,代謝體學, | zh_TW |
| dc.subject.keyword | Type 2 diabetes,hepatic cell line,islet beta cell line,wild bitter gourd (Momordica Charantia),metabolomics, | en |
| dc.relation.page | 93 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-07-25 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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