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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃青真 | |
dc.contributor.author | Meng-Ting Wu | en |
dc.contributor.author | 吳夢婷 | zh_TW |
dc.date.accessioned | 2021-06-13T15:45:18Z | - |
dc.date.available | 2009-07-07 | |
dc.date.copyright | 2008-07-07 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-07-02 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37816 | - |
dc.description.abstract | 停經婦女因雌激素分泌減少,常伴隨有熱潮紅、盜汗、心悸等通稱更年期症候群,且心血管疾病、骨質疏鬆症、老人失智症等疾病的風險亦提高。文獻指出齧齒類動物於卵巢剔除後會產生與停經婦女相似的症狀,如肥胖、骨質疏鬆及代謝症候群等,但在雌激素的補充後可有效地改善這些症狀。本實驗初步篩選評估七種傳統婦科常用草藥萃取物:甘草 EA、甘草 MeOH、何首烏 MeOH、當歸 MeOH、桃仁 MeOH、熟地黃 EA 及白朮 EA 萃物之雌激素效應。並進一步利用卵巢剔除小鼠探討具植物雌激素活性的生、熟山藥及生山藥、苜蓿芽與白朮之 EA 萃物在 in vivo 之效應。
第一部份實驗先以 CHO-K1 細胞共同轉染 chimeric receptor of GAL4-hERα (or β) LBD 及 (UAS)4-tk-alkaline phosphatase reporter 分析活化 ERα 與 ERβ 之能力。接著利用人類乳癌細胞株 MCF-7 及子宮內膜癌細胞株 Ishikawa 增生試驗 (MTT 試驗),評估各萃物對雌激素依賴型癌細胞之增生影響。並測試樣品對 Ishikawa 細胞中,內源性 ER 下游基因 ALP 活性之影響。藉由以上三種系統,評估各樣品萃物之雌激素效應。 結果顯示,除當歸 MeOH 選擇性活化 ERα,桃仁 MeOH 萃物對 ERα 及 ERβ 活化程度相當,其餘五種萃物活化 ERβ 的能力大於 ERα。其中又以熟地黃 EA 與白朮 EA 萃物的活化效果最高。於 MCF-7 增生試驗中,桃仁 MeOH 及熟地黃 EA 萃物顯著降低 MTT 染色值,而甘草 EA、何首烏 MeOH 及白朮 EA 萃物則是顯著增加 MTT 染色值。而 Ishikawa 細胞增生試驗中,甘草 MeOH (低劑量)、熟地黃 EA 及白朮 EA 萃物顯著降低 MTT 染色值,甘草 EA、甘草 MeOH (高劑量)、何首烏 MeOH 及桃仁 MeOH 萃物則是顯著增加 MTT 染色值。而在 Ishikawa 細胞中對 ALP 活性影響試驗中,所有萃物均可顯著增加 ALP 活性。綜合轉染、癌細胞增生及內源性 ER 下游 ALP 酵素活性分析,以熟地黃 EA 及白朮 EA 萃物最具開發潛力。前者活化 ERβ 優於 ERα,並可抑制 MCF-7 及 Ishikawa 細胞之增生。後者為轉染試驗中活性最高者,且活化 ERβ 效果亦優於 ERα。 進一步利用卵巢剔除鼠,探討本研究室以純化分離出植物雌激素活性成分之生山藥 EA 萃物 (台農二號) 及苜蓿芽 EA 萃物,以及生、熟山藥粉和第一部份實驗篩選出 ER 活性最高之白朮 EA 萃物,在 in vivo 之效應。 實驗採用 C57BL/6J 品系母鼠,於 8 週齡進行偽手術 (Sham) 或卵巢剔除手術 (OVX),術後第二天 OVX 鼠依體重隨機分為 B (Basal)、RY (生山藥)、CY (熟山藥)、RYEA (生山藥 EA 萃物)、AEA (苜蓿芽 EA 萃物)、AM (白朮 EA 萃物) 及 E2 (2 mg 17β-estradiol/kg diet) 組,Sham 鼠餵食 basal diet (SB 組),餵食實驗飼料 14 週後犧牲。 結果顯示 B 組的確因卵巢剔除而產生肥胖、血糖異常及胰島素抗性等代謝症候群之症狀。而 E2、RY、CY 及 AM 具有顯著改善以上情況之效果:E2 及 RY 可顯著改善體重、飼料/能量利用率、血糖、肝臟三酸甘油酯 (TG)、子宮周圍 (PWAT) 及腎周圍脂肪 (RWAT) 相對重量與胰島素抗性 (p <0.05);RY 組的血清及肝臟中脂質含量更顯著低於 B 組 (p <0.05);CY 組 RWAT 重、血脂及胰島素抗性顯著較 B 組低 (p <0.05);AM 組體重、血糖及胰島素抗性指標 (HOMA-IR index) 顯著低於 B 組 (p <0.05),可能與攝食量顯著較低有關 (p <0.05)。但在本研究中,AEA 及 RYEA 並無改善代謝症候群之效果。 鈣平衡試驗結果顯示餵食 3 週後,各樣品 (RY、CY、AEA、RYEA 及 AM) 顯著增加鈣的吸收保留 (p<0.05)。但到 14 週時,只剩 RY 仍具此效果,而 AEA、RYEA 及AM 組反而顯著低於 B 組 (p <0.05),E2 對鈣平衡則無影響。 進一步分析股骨及腰椎骨後三節中的灰份及鈣含量,股骨分析結果,E2 組的乾重、灰份、鈣量及骨鈣/乾重之比例皆顯著高於 B 組 (p <0.05)。RY 組的股骨乾重及灰份重雖顯著低於 B 組 (p <0.05),但鈣/灰份比例則顯著高於 B 組 (p <0.05)。AEA組的鈣量、鈣/乾重及鈣/灰份重之比例顯著高於 B 組 (p <0.05)。AM 組的乾重、灰份重、鈣量、鈣/乾重及鈣/灰份重之比例顯著低於 B 組 (p<0.05)。而在腰椎骨後三節之分析結果顯示,B 組的鈣/乾重比例顯著低於 SB 組 (p<0.05)。E2 組的灰份、鈣量及骨鈣/乾重之比例皆顯著高於 B 組 (p <0.05)。RY 組的灰份、鈣量顯著低於 B 組 (p <0.05)。CY 及 AEA 組的鈣/灰份重比例顯著高於 B 組 (p <0.05)。RYEA 組的鈣/乾重及鈣/灰份重比例顯著高於 B 組 (p<0.05)。AM 組的乾重及灰份重顯著低於 B組,但鈣/乾重及鈣/灰份重之比例顯著高於 B 組 (p<0.05)。 文獻指出雌激素受器與過氧化體增殖受器 α 會互相影響,即之間具有 cross-talk 現象,而 RY 組攝食量高於其他各組,但具有最好的改善效果,是否影響脂質吸收或與脂質代謝相關酵素?發現 RY 並未影響參與脂質代謝的 PPARα 下游基因 Acyl-CoA oxidase (ACO) 酵素活性,而是抑制脂質吸收。 進一步以 Real-time QPCR 分析子宮、肝臟及子宮周圍脂肪組織中 ER 下游基因 Brain isozyme of creatine kinase (Ckb) 及子宮中 ER 下游基因 Progesterone receptor (Pgr) 之 mRNA 表現量。子宮中 Ckb 表現量以 RY、AEA 及 RYEA 組顯著高於 B 組 (p<0.05),肝臟中 Ckb 表現量以 E2 組顯著高於 B 組 (p<0.05)。其餘則是各組間與 B 組無顯著差異。 綜此,本研究篩選出七種具植物雌激素活性之中藥萃物,其中以熟地黃 EA與白朮 EA 萃物最具開發潛力。生、熟山藥可改善卵巢剔除鼠之代謝症候群,而苜蓿芽、生山藥及白朮 EA 萃物對骨鈣有部分助益,具有婦女於更年期及停經後保健食品發展之潛力。而其改善代謝症候群之機制,除已知的雌激素活性,似乎亦透過其他途徑,值得繼續探討。 | zh_TW |
dc.description.abstract | Menopausal and post-menopausal women are known to have higher risk of metabolic syndrome, cardiovascular disease and osteoporosis as a result of estrogen deficiency. Estrogen administration reduces adipose tissue mass in both human and animals. Phytoestrogens are considered to be a safer alternative for HRT for post-menopausal healthcare. Estrogenic activity is known to be mediated by estrogen receptors (ER). To test ER agonistic/antagonistic activity, a transactivation assay using CHO-K1 cells transiently co-transfected with a (UAS)4-tk-alkaline phosphatase reporter and a chimeric receptor of GAL4-hERα (or β) LBD was used to screening a series of herbs commonly used in the formulations of gynecological remedies in traditional Chinese medicine. Herbal materials were extracted by methanol or ethyl acetate (EA). It was found that 7 extracts from 6 Chinese herbs, Glycyrrhiza inflate (GI), Polygonum multiflorum (PM), Angelica sinensis (AS), Prunus persica (PP), Rehmannia glutinosa (RG), and Atractylodes macrocephala (AM), activated ERα or ERβ or both. Except for methanol extract of AS and PP that activated ERα selectively or ERα and ERβ to a similar extent, all the remaining 5 herb extracts exhibited higher activity on ERβ than on ERα. In ERα/β antagonistic activity assay, all extracts exhibited synergistic effects. The 7 herbal extracts were then assessed for the effect on proliferation of human breast cancer line MCF-7 and endometrial cancer cell line Ishikawa by MTT assay in the absence or presence of 1 nM 17β-estradiol (E2). AS and RG extracts significantly reduced the MTT value of MCF-7 cells. Methanol extract of GI and EA extracts of RG and AM significantly reduced MTT value of Ishikawa cells. To test the sample extracts of ER target gene expression, treated Ishikawa cells were assayed for the ALP activity. All 7 extracts increased ALP activity of Ishikawa cells. Among the 7 samples, RG extract exhibited significant estrogenic activity in both transactivation assay and Ishikawa cell ALP assay, but did not increased proliferation of MCF-7 and Ishikawa cell. AM extract showed highest ER transactivation activity but significantly increased proliferation of MCF-7 cell and significantly reduced proliferation of Ishikawa cell.
To assess the effects of these phytoestrogen material in vivo, C57BL/6J mice were Sham-operated or Ovariectomized (OVX) at 8 weeks of age. Two days after operation, they were assigned to various diet groups. Sham mice were fed the basal diet (SB) and OVX mice were respectively fed the basal diet(B), diets containing E2 (2 mg 17β-estradiol/ kg diet), RY (raw yam), CY (cooked yam), EA extract of alfalfa (AEA), RY (RYEA) or AM (Atractylodes macrocephala) for 14 weeks. Mice fed E2, RY, CY and AM diets were less obese, had improved hyperglycemia and insulin resistance induced by ovariectomy. Compared to the OVX B group, the E2 and RY OVX mice had significantly lower body weight, feed/energy efficiency, serum glucose, hepatic triglyceride, glucose intolerance, as well as parametrial (PWAT) and retroperitoneal (RWAT) white adipose tissue weight (p<0.05). In addition, the RY group also had lowered cholesterol in both serum and liver; the CY group had improved dyslipidemia and insulin resistance and less RWAT weight; the AM group had less body weight, serum glucose and HOMA-IR index. An inhibited fat absorption was observed in only the RY group. Results of calcium balance experiment indicated that RY, CY, AEA, RYEA and AM increased calcium absorption and retention after 3 weeks of feeding, but only RY had the prolonged effect. E2 and AEA groups were found to have higher femur calcium compared to the remaining groups (p<0.05). To examine the effect of the dietary treatment on mRNA expression of ER target genes, brain isozyme of creatine kinase (Ckb) and progesterone receptor (Pgr), in uterus, liver and parametrial white adipose tissue were analyzed using real-time RT-PCR. Results showed that OVX mice receiving the RY, AEA and RYEA diets showed increased Ckb expression in uterus. Only E2 group had higher Ckb expression in liver (p<0.05). In conclusion, 7 herbal extracts were demonstrated to have phytoestrogenic activity. Rehmannia glutinosa and Atractylodes macrocephala might have highest potential to be developed as functional foods for post-menopausal women. Raw yam diet can ameliorate most risk factors of the metabolic syndrome in OVX mice model, other phytoestrogenic diet treatments also improved some, but not all risk factors. AEA, RYEA and AM had partial benefits on bone calcium. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T15:45:18Z (GMT). No. of bitstreams: 1 ntu-97-R95b47303-1.pdf: 1759633 bytes, checksum: 8b4a2d92fd7246e4d27756575189cde4 (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | 論文口試委員審定書.………………………………………………………… i
謝誌……………………………………………………………………………… iii 中文摘要………………………………………………………………………… vii 英文摘要……………………………………………………………………… xi 縮寫對照表……………………………………………………………………… xv 第一章 緒論…………………………………………………………………… 1 第一節 前言……………………………………………………………… 1 第二節 文獻回顧………………………………………………………… 1 一、雌激素受器 (Estrogen receptor, ER) ……………………………… 1 (一) ER 的種類………………………………………………… 1 (二) ER 的分佈………………………………………………… 3 (三) ER 的作用機制與下游基因……………………………… 4 (四) ER 剔除鼠………………………………………………… 4 二、植物雌激素 (Phytoestrogen, PE) ………………………………… 8 三、SERM……………………………………………………………… 10 四、雌激素缺乏與代謝症候群………………………………………… 11 (一) 代謝症候群之定義………………………………………… 11 (二) 雌激素/植物雌激素與代謝症候群……………………… 13 五、雌激素缺乏與鈣質代謝…………………………………………… 14 (一) 鈣質消化、吸收、運輸及排出……………………………… 14 (二) 雌激素及植物雌激素對鈣質代謝之影響………………… 15 六、植物雌激素活性材料……………………………………………… 16 (一) 甘草 (Glycyrrhiza inflate Bat) …………………………… 16 (二) 何首烏 (Polygonum multiflorum Thunb.) ………………… 17 (三) 當歸 (Angelica sinensis (Oliv.) Diels) …………………… 17 (四) 桃仁 (Prunus persica (L.) Batsch) ……………………… 17 (五) 熟地黃 (Rehmannia glutinosa Libosch.) ………………… 18 (六) 白朮 (Atractylodes macrocephala koidz.) ……………… 18 (七) 山藥………………………………………………………… 19 (八) 苜蓿芽……………………………………………………… 19 第三節 研究假說與架構………………………………………………… 19 一、研究假說………………………………………………………… 19 二、實驗架構………………………………………………………… 20 第二章 傳統中醫常用婦科草藥之雌激素效應……………………………… 21 第一節 前言……………………………………………………………… 21 第二節 材料與方法……………………………………………………… 22 一、試劑與器材……………………………………………………… 22 (一) 細胞株……………………………………………………… 22 (二) 細胞培養基………………………………………………… 22 (三) 血清………………………………………………………… 23 (四) 細胞用試劑………………………………………………… 23 (五) 短暫轉染用質體…………………………………………… 24 (六) 短暫轉染用試劑…………………………………………… 24 (七) 分析 Alkaline phosphatase (ALP) 之試劑與器材…… 25 (八) 藥材樣品及標準品………………………………………… 25 (九) 細胞培養用器材…………………………………………… 26 二、實驗方法………………………………………………………… 27 (一) 細胞培養方法……………………………………………… 27 (二) 短暫轉染試驗……………………………………………… 28 (三) MCF-7 細胞增生試驗…………………………………… 28 (四) ALP活性分析……………………………………………… 29 (五) 細胞存活率之分析- MTT染色法………………………… 30 (六) 統計分析…………………………………………………… 30 第三節 結果……………………………………………………………… 31 一、各藥材萃物之 ERα 活性……………………………………… 31 二、各藥材萃物之 ERβ 活性……………………………………… 31 三、各藥材萃物對 MCF-7 細胞株增生之影響…………………… 32 四、各藥材萃物對 Ishikawa 細胞株 ALP 活性之影響…………… 33 五、各藥材萃物對 Ishikawa 細胞株增生之影響…………………… 34 第四節 討論與結論……………………………………………………… 41 一、甘草 EA 及 MeOH 萃物之總雌激素效應…………………… 41 二、何首烏 MeOH 萃物之總雌激素效應………………………… 42 三、當歸 MeOH 萃物之總雌激素效應…………………………… 42 四、桃仁 MeOH 萃物之總雌激素效應…………………………… 43 五、熟地黃 EA 萃物之總雌激素效應……………………………… 43 六、白朮 EA 萃物之總雌激素效應………………………………… 44 七、結論……………………………………………………………… 44 第三章 植物雌激素活性材料於卵巢剔除鼠之效應………………………… 47 第一節 前言……………………………………………………………… 47 第二節 材料與方法……………………………………………………… 48 一、實驗大綱………………………………………………………… 48 二、動物飼養及手術…………………………………………………… 49 三、飼料……………………………………………………………… 49 (一) 山藥粉……………………………………………………… 49 (二) 苜蓿芽、生山藥粉及白朮乙酸乙酯萃物………………… 50 (三) 飼料配製…………………………………………………… 50 四、口服葡萄糖耐受性試驗………………………………………… 50 (一) 藥品配製…………………………………………………… 50 (二) 實驗流程…………………………………………………… 51 (三) 血清葡萄糖分析…………………………………………… 51 (四) 血清胰島素分析…………………………………………… 51 五、動物犧牲………………………………………………………… 52 六、血清分析………………………………………………………… 55 (一) 葡萄糖分析………………………………………………… 55 (二) 三酸甘油酯分析…………………………………………… 55 (三) 膽固醇分析………………………………………………… 55 (四) 胰島素分析………………………………………………… 55 (五) 鈣質分析…………………………………………………… 56 七、胰島素抗性指標 HOMA-IR index 計算……………………… 56 八、肝脂分析………………………………………………………… 56 (一) 肝脂質萃取液製備………………………………………… 56 (二) 肝臟三酸甘油酯含量測定………………………………… 56 (三) 肝臟膽固醇含量測定……………………………………… 57 九、脂質吸收試驗…………………………………………………… 57 (一) 實驗流程…………………………………………………… 57 (二) 飼料及糞便粗脂肪含量分析……………………………… 57 (三) 脂質吸收計算……………………………………………… 57 十、鈣平衡試驗……………………………………………………… 58 (一) 飼料及糞便灰份分析……………………………………… 58 (二) 藥品配製…………………………………………………… 58 (三) 飼料及糞便鈣含量分析…………………………………… 58 (四) 尿鈣含量分析……………………………………………… 59 (五) 鈣吸收狀況計算…………………………………………… 59 十一、肝臟過氧化 ACO 酵素活性分析…………………………… 59 (一) 原理………………………………………………………… 59 (二) 藥品配製…………………………………………………… 60 (三) 去核上清液之製備………………………………………… 62 (四) 去核上清液蛋白質含量測定……………………………… 63 (五) 活性分析…………………………………………………… 63 (六) 酵素活性計算……………………………………………… 63 十二、骨頭分析……………………………………………………… 64 (一) 藥品配製…………………………………………………… 64 (二) 組織切片染色……………………………………………… 65 (三) 骨頭乾重、灰份及鈣含量分析……………………………… 65 十三、Real-time PCR 法分析子宮、肝臟及子宮周圍脂肪中 ER 下游基因 mRNA 表現量………………………………………… 66 (一) 總 RNA 之抽取…………………………………………… 66 (二) 將總 RNA 反轉錄成 cDNA…………………………… 67 (三) Real-time PCR 分析 ER 下游基因表現………………… 67 十四、統計分析……………………………………………………… 68 第三節 結果……………………………………………………………… 69 一、體重變化、攝食量及飼料利用率………………………………… 69 二、組織器官相對重量……………………………………………… 70 三、血清分析結果…………………………………………………… 71 (一) 血清葡萄糖………………………………………………… 71 (二) 血清三酸甘油酯…………………………………………… 71 (三) 血清膽固醇………………………………………………… 72 (四) 血清鈣分析………………………………………………… 72 (五) 口服葡萄糖耐受性試驗…………………………………… 72 四、胰島素抗性指標 HOMA-IR index……………………………… 73 五、肝臟脂質分析…………………………………………………… 73 (一) 三酸甘油酯………………………………………………… 73 (二) 膽固醇……………………………………………………… 74 六、肝臟過氧化體 ACO 酵素活性………………………………… 74 七、脂質及鈣平衡試驗結果………………………………………… 74 (一) 各組飼料脂質及鈣質含量………………………………… 74 (二) 每日攝食量、糞便排出量及糞便含水率………………… 74 (三) 脂質攝取與吸收狀況……………………………………… 75 八、骨頭分析………………………………………………………… 77 (一) 重量………………………………………………………… 77 (二) 灰份分析…………………………………………………… 77 (三) 骨鈣分析…………………………………………………… 78 (四) 股骨橫切面圖……………………………………………… 79 九、子宮、肝臟及子宮周圍組織中 ER 下游基因 Ckb 及 Pgr 之 mRNA 表現量…………………………………………………… 79 (一) 子宮………………………………………………………… 79 (二) 肝臟………………………………………………………… 79 (三) 子宮周圍脂肪……………………………………………… 79 第四節 討論與結論……………………………………………………… 106 一、卵巢剔除小鼠探討代謝症候群之動物模式確立……………… 106 二、卵巢剔除、偽手術及雌激素補充對鈣質代謝及骨鈣之影響…… 107 三、雌激素活性樣品對子宮之影響………………………………… 108 四、各植物雌激素樣品與E2 對卵巢剔除鼠生理狀況影響……… 109 (一) 生山藥、熟山藥及生山藥 EA 萃物……………………… 109 (二) 苜蓿芽及白朮 EA 萃物………………………………… 111 五、影響途徑………………………………………………………… 112 六、結論……………………………………………………………… 113 第四章 綜合討論與總結論…………………………………………………… 117 第一節 綜合討論………………………………………………………… 117 一、熟地黃及白朮乙酸乙酯萃物選擇性活化 ERβ 且某些情況可抑制癌細胞增生…………………………………………………… 117 二、生、熟山藥粉可改善卵巢剔除鼠之代謝症候群,而生山藥粉乙酸乙酯萃取物可增加骨鈣含量………………………………… 119 第二節 總結論…………………………………………………………… 121 第五章 參考文獻……………………………………………………………… 123 附錄……………………………………………………………………………… 139 一、CHO-K1 細胞對七種中藥萃物樣品之劑量耐受度………………… 139 二、CHO-K1 細胞經共同轉染 pBKCMV GAL4-hERα/β 與 pBKCMV(UAS)4-ALP 後對 E2、Genistein 及 Daidzein 之劑量反應曲線…………………………………………………………………………… 140 | |
dc.language.iso | zh-TW | |
dc.title | 數種具植物雌激素活性材料對卵巢剔除鼠代謝症候群及鈣代謝之影響 | zh_TW |
dc.title | Effects of Phytoestrogenic Foods/Herbs on Metabolic Syndrome and Ca Metabolism in Ovariectomized Mice | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳曉鈴,吳文惠,蘇南維,林璧鳳 | |
dc.subject.keyword | 植物雌激素,雌激素受器,代謝症候群,骨質,山藥, | zh_TW |
dc.subject.keyword | Phytoestrogen,Estrogen receptor,Metabolic syndrome,Bone,Yam, | en |
dc.relation.page | 141 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2008-07-02 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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