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研究生:吳沛承
研究生(外文):Pei-Cheng Wu
論文名稱:探討CPR (cytopiloyne receptor) 與骨化相關因子以及植物化合物之間的關係
論文名稱(外文):CPR (cytopiloyne receptor): relationship between osteogenic factors and phytocompounds
指導教授:張力天
指導教授(外文):Lee-Tian Chang
口試委員:陳建榮梁熾龍
口試委員(外文):Jeng-Rung ChenChih-Lung Liang
口試日期:2017-07-07
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:39
中文關鍵詞:骨質疏鬆;大花咸豐草;聚多炔糖苷
外文關鍵詞:Osteoporosis;Bidens pilosa;cytopiloyne
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骨質疏鬆為目前最常見的骨病,骨質疏鬆分為兩類1. 原發性骨質疏鬆(Primary osteoporosis):是以骨量減少、骨的微觀結構退化為特徵的,致使骨的脆性增加以及易於發生骨折的一種全身性骨骼疾病。2. 繼發性骨質疏鬆(Secondary osteoporosis):是由於疾病或藥物等原因所致的骨量減少、骨微結構(Microarchitectural deterioration)破壞、骨脆性增加和易於骨折的代謝性骨病。有證據顯示影響骨質疏鬆的機制主要是由破骨細胞(Osteoclast)以及成骨細胞(Osteoblast)之間的交互協調作用。實驗室先前利用大花咸豐草(Bidens pilosa)的聚多炔糖苷(Cytopiloyne, CP)針對 Peroxisome proliferator-activated receptor-γ (PPARγ;過氧化物酶體增殖物活化受體γ)具有調節3T3-L1脂肪細胞的抑制作用,CP抑制3T3-L1脂肪細胞的PPARγ以及CCAAT/enhancer binding proteins(C/EBPs;CCAAT增強子結合蛋白家族等脂肪因子)的表現,另外也利用CPR基因剔除db/db小鼠證明CP會促進二型糖尿病小鼠胰島細胞生長以及抑制β胰島細胞凋亡。目前本實驗除了研究CP與骨化相關因子Runt-related transcription factor 2(RUNX2;Runt相關轉錄因子)以及Alkaline phosphaste(ALP;鹼性磷酸酶)的關係之外,也利用可以促進胰島β細胞功能、抑制脂肪細胞分化及促進骨化細胞的分化的八種天然純物質分別測試於MC3T3-E1骨母細胞,確定分別對於MC3T3-E1骨母細胞細胞中的CPR影響,以此作為一個平臺來篩選其他可能改善骨質疏鬆的藥物,PCR結果顯示其中的Kaempferol是影響CPR基因與蛋白質表現最具潛力的天然純物質。
Osteoporosis is the most common bone disease. There are two categories of osteoporosis. Primary osteoporosis is the systemic skeletal disease which characterised by bone loss, bone microstructure degradation resulting in bone brittleness increased and lead fracture. Secondary osteoporosis is the metabolic bone disease which due to bone loss, bone microstructure damage, bone fragility increased and easy to fracture by other disease or drugs. The evidence showed that the mechanism of osteoporosis is mediated by the interaction between osteoblasts and osteoblasts. Our laboratory previously confirmed the cytopiloyne (CP) which extract by Bidens pilosa regulated the factor of adipogenesis activity of inhibition as peroxisome proliferator-activated receptor-γ (PPARγ) in 3T3-L1 adipocyte. Besides, we also proved that CP would promote the growth of islet cells in type 2 diabetic mice and inhibit β-cell apoptosis by CP receptor knockout db/db mice. In this study, we observed the relationship between CPR and the facties of oeteogenesis as Runt-related transcription factor 2 (RUNX2) and Alkaline phosphaste (ALP). We also used the other eight phytocompounds to test the effects of MC3T3-E1 cells with regard to the influence of CPR, which could be a platform to screen out the better drugs than CP. Finally, the PCR results showed that kaempferol is the most potential effect drug in expression of protein and gene.
中文摘要…………………………………………………………………………………………………………… i
Abstract……………………………………………………………………………………………………… ii
目次………………………………………………………………………………………………………………… iii
圖目次……………………………………………………………………………………………………………… v
第一章 緒言……………………………………………………………………………………………… 1
第二章 文獻探討……………………………………………………………………………………… 3
第一節 骨質疏鬆的分類……………………………………………………………………………… 3
一、原發性骨質疏鬆(Primary osteoporosis)…………………………… 3
二、繼發性骨質疏鬆(Secondary osteoporosis)……………………… 3
第二節 糖尿病引起的骨質疏鬆………………………………………………………………… 3
第三節 骨質疏鬆治療…………………………………………………………………………………… 4
第四節 骨質影響因子…………………………………………………………………………………… 5
一、脂化作用(Adipogenesis)……………………………………………………………… 5
二、骨化作用(Osteogenesis)……………………………………………………………… 5
三、Wnt signaling pathway…………………………………………………………… 6
第五節 植物萃取純化物……………………………………………………………………………… 6
一、Cytopiloyne……………………………………………………………………………………… 6
二、Kaempferol………………………………………………………………………………………… 7
三、Curcumin……………………………………………………………………………………………… 7
四、Berberine…………………………………………………………………………………………… 7
五、Emodin…………………………………………………………………………………………………… 8
六、Resveratrol……………………………………………………………………………………… 8
七、Isopsoralen……………………………………………………………………………………… 8
八、Quercetin…………………………………………………………………………………………… 9
九、Puerarin……………………………………………………………………………………………… 9
第三章 材料與方法………………………………………………………………………………… 10
第一節 實驗動物…………………………………………………………………………………………… 10
第二節 成骨細胞株培養……………………………………………………………………………… 10
第三節 逆轉式聚合酶鏈鎖反應………………………………………………………………… 10
一、材料準備…………………………………………………………………………………………………… 10
二、實驗步驟…………………………………………………………………………………………………… 11
第四節 蛋白質萃取與西方墨點法……………………………………………………………… 11
第五節 植物萃取純化物……………………………………………………………………………… 11
第六節 胰島素酵素活性試驗……………………………………………………………………… 12
一、材料準備及實驗步驟……………………………………………………………………………… 12
第四章 實驗結果…………………………………………………………………………………………… 13
第一節 利用RT-PCR鑑定C57BL/6小鼠全組織CPR的表現……………… 13
第二節 MC3T3-E1細胞分化及PCR鑑定………………………………………………… 13
第三節 MC3T3-E1 CPR KO細胞分化及PCR鑑定……………………………… 13
第四節 九種天然化合物之CPR表現以西方墨點法以及PCR鑑定……… 14
第五節 Insulin reduction assay………………………………………………… 14
第五章 討論…………………………………………………………………………………………………… 21
第六章 參考文獻…………………………………………………………………………………………… 24
附圖…………………………………………………………………………………………………………………… 36
縮寫表………………………………………………………………………………………………………………… 39
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