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研究生: 田秉玉
Tien, Ping-Yu
論文名稱: 臺灣天仙果萃取物對人類腸道上皮細胞株Caco-2鈣離子運輸與糖尿病去卵巢雌性小鼠骨質疏鬆之影響
Effects of Ficus formosana extract on calcium transport into human intestinal epithelial Caco-2 cells and osteoporosis in diabetic ovariectomized female mice
指導教授: 沈賜川
Shen, Szu-Chuan
吳瑞碧
Wu, Swi-Bea
丁俞文
Ting, Yu-Wen
學位類別: 碩士
Master
系所名稱: 營養科學碩士學位學程
Graduate Program of Nutrition Science
論文出版年: 2020
畢業學年度: 108
語文別: 中文
論文頁數: 80
中文關鍵詞: 骨質疏鬆症臺灣天仙果Caco-2細胞糖尿病去卵巢小鼠鈣離子轉運
英文關鍵詞: osteoporosis, Ficus formosana, Caco-2 cells, diabetic ovariectomized female mice, calcium transportation
DOI URL: http://doi.org/10.6345/NTNU202001369
論文種類: 學術論文
相關次數: 點閱:195下載:0
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  • 隨著人口急遽老化,骨質疏鬆症的盛行率也正持續攀升。骨質疏鬆症為臨床常見疾病,不僅會導致骨質流失最終造成患者骨質密度減弱,也會增加骨折的機率。臺灣天仙果 (Ficus formosana Maxim.) 為桑科榕屬植物,是民間普遍使用之食補藥材,具有促進骨骼生長及活血化瘀等功效。本研究利用人類大腸癌細胞株Caco-2細胞transwell seeding單層膜模式評估天仙果莖部熱水萃取物對腸道細胞促鈣轉運之效果,並且以糖尿病去卵巢小鼠動物模式評估其延緩骨質疏鬆之效果及可能機制。首先利用MTT assay測試臺灣天仙果莖部熱水萃取物之毒性,再以Caco-2細胞模擬腸道鈣離子轉運的細胞模式,於transwell上室同時加入鈣離子與天仙果萃取物後,分別在不同時間點抽取下室液,並以 Arsenazo Ⅲ為呈色劑利用分光光譜法測定鈣離子濃度。MTT細胞存活率測試結果顯示,天仙果萃取物於濃度25-500 ppm時對Caco-2細胞並無毒性產生。添加高低濃度(250-50 ppm)的天仙果莖部熱水萃取物處理,可明顯提升Caco-2腸道細胞的鈣離子轉運能力(p<0.05)及加腸道鈣轉運相關蛋白質Claudin 2、Claudin 12、TRPV6、PMCA1和CaBP-9k表現量(p<0.05),且也增加與維生素D調控鈣離子途徑相關蛋白VDR之表現與抑制CYP24A1降解1,25(OH)2D3的能力。在去卵巢之糖尿病雌性小鼠餵食含有高劑量天仙果莖部熱水萃取物之飼料6週後,其血清骨鈣素(Osteocalcin)顯著增加,而骨膠原蛋白碳末端肽鏈(C-telopeptide of type I collagen CTX-1)則顯著降低(p<0.05);另一方面,雖然骨密度(Bone mineral density)各組間無顯著差異,但是臺灣天仙果莖部熱水萃取物處理組之骨小樑的厚度與數目皆較去卵巢糖尿病小鼠為高,顯示具有延緩骨質酥鬆之效果。本研究結果可作為評估臺灣天仙果植株莖部開發骨骼保健膳食補充劑或食品時之參考。

    Increasing calcium absorption is the most important and well known to prevent and cure osteoporosis in human been. Ficus formosana (FF), belongs to Moraceac Ficus genus, is a perennial evergreen shrub. The Ficus formosana has been used as folk medicine to cure osteoporosis for decades due to its ability on enhancing human bone growth and adjust menstruation ect. This study evaluates the effects of Ficus formosana roots hot water extract (FFE) on calcium absorption in human intestinal epithelial Caco-2 cells and anti-osteoporosis in diabetic ovariectomized female mice. The cell viabilities of Caco-2 cells were measured by MTT assay. The apical side of the transwell insert was used to assess the effect of FFE on calcium transport across Caco-2 monolayers. HBSS buffer was collected from the basolateral side and analyzed by Arsenazo Ⅲ to measure calcium ion concentration. The results show that FFE at the tested ranges of 25-250 ppm exhibited no cellular cytotoxicity in Caco-2 cells. The treatment of FFE revealed a significantly increase the total calcium transported across Caco-2 monolayers. Western blot analysis shows FFE increased Ca2+ absorption-related proteins includes Claudin 2, Claudin 12, TRPV6, PMCA1b and Calbindin-D(9K) in the Caco-2 cells (p<0.05).Also, FFE increased Vitamin D-inducible calcium transport related proteins VDR, and inhibited CYP24A1. In addition, treatment of FFE for 6 weeks significantly increased serum osteocalcin, whereas decrease serum C-terminal telopeptides type I collagen compared with diabetic ovariectomized female mice. Moreover, FFE increased trabecular thickness and numbers in diabetic ovariectomized female mice. The above observations suggest that FFE possesses potential as a material to develop nutraceuticals or dietary supplements on improving bone health.

    第一章、前言 1 第二章、文獻回顧 2 第一節、骨頭之結構與生化代謝 2 一、骨骼之組成 2 二、骨質重塑循環 3 第二節、骨質疏鬆症(Osteoporosis) 4 一、骨質疏鬆症定義與流行病學 4 二、骨質疏鬆症之診斷與追蹤 4 三、骨質疏鬆症之風險因子 8 四、骨質疏鬆症之預防 9 第三節、糖尿病與骨質 10 一、 糖尿病定義、流行病學與其合併症 10 二、糖尿病之主要類型 10 三、糖尿病與骨質疏鬆症之關係 11 四、糖尿病患者骨質疏鬆症之治療與預防 13 第四節、人體中鈣質吸收與調節 14 一、鈣對人體之重要性 14 二、鈣吸收之種類與其機制 15 第五節、臺灣天仙果 18 一、臺灣天仙果 18 二、臺灣天仙果之藥理作用 18 第三章、研究動機與目的及實驗架構 19 第一節、研究動機與目的 19 第二節、實驗架構 20 第四章、實驗材料與方法 21 第一節 實驗藥品與儀器 21 一、實驗藥品與試劑 21 二、儀器設備 24 第二節、樣品製備之材料與方法、與總酚、類黃酮類和 Umbelliferone 含量分析 26 一、實驗樣品來源 26 二、實驗方法 26 三、總多酚類含量測定(Total Phenolic Content) 26 四、總類黃酮含量測定(Total Flavonoid Content) 26 三、Umbelliferone 含量分析 27 第三節、細胞實驗之材料與方法 28 一、實驗細胞株 28 二、實驗方法 28 第三節、動物試驗設計 37 一、實驗動物 37 二、實驗動物飼料 37 三、實驗動物誘導與分組 38 四、實驗方法 39 第五章、結果 41 第一節、臺灣天仙果莖部熱水萃取物製備與Umbelliferone含量 41 一、臺灣天仙果莖部熱水萃取物製備 41 二、總多酚類與總類黃酮含量測定 41 三、Umbelliferone含量測定試驗 41 第二節、臺灣天仙果莖部熱水萃取物對Caco-2細胞株存活率與鈣離子轉運之影響 46 一、臺灣天仙果莖部熱水萃取物對Caco-2細胞株存活率之影響 46 二、跨上皮細胞電阻值(Transepithelial electrical resistance, TEER) 47 三、臺灣天仙果莖部熱水萃取物對Caco-2細胞株鈣轉運之影響 48 四、臺灣天仙果莖部熱水萃取物對促進腸道細胞株Caco-2鈣離子轉運作用機制之探討 49 第三節、臺灣天仙果莖部熱水萃取物對糖尿病去卵巢雌性小鼠糖尿病之影響 56 一、體重與飲水、食物攝取量 56 二、血液血糖、胰島素值 58 三、糖尿病相關之血液生化值 58 第四節、臺灣天仙果熱水萃取物對糖尿病去卵巢雌性小鼠骨質疏鬆之影響 61 一、骨質代謝相關之血液生化值 61 二、Micro-CT 分析 64 第六章、討論 69 第一節、臺灣天仙果莖部熱水萃取物對腸道細胞株之鈣吸收之影響與機制探討 69 一、臺灣天仙果莖部熱水萃取物促進腸道細胞株之鈣轉運 69 二、臺灣天仙果莖部熱水萃取物促進腸道細胞株鈣轉運作用機制之探討 69 第二節、臺灣天仙果對去卵巢糖尿病雌鼠之骨質結構與生化代謝指標之探討 71 一、臺灣天仙果對血鈣與血磷之影響 71 二、臺灣天仙果對骨質重塑作用之影響 72 三、臺灣天仙果促進骨質結構之探討 73 第七章、結論 74 參考文獻 75

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