攝護腺癌全世界中，具有相當高的發生率以及死亡率，而在台灣攝護腺癌的發生率逐年上升且居高不下的死亡率。目前許多研究指出，攝護腺癌的發生及惡化與雄性激素和雄性激素受體(Androgen receptor, AR)的表現量有密切的關係；甚至有研究表示，AR也可加速雄激素非依賴性攝護腺癌的癌細胞生長，也與攝護腺癌復發和轉移特性有關。因此抑制雄性激素與AR是治療攝護腺癌的方式之一。本實驗室在過去發表的文獻中提到，AR Ser 81位點磷酸化對於AR蛋白質的穩定性相當重要。Metformin主要用於治療糖尿病的藥物，可經由抑制攝護腺癌重要生長指標AR mRNA的表現進而影響AR的訊息傳遞路徑。許多研究發現，Metformin可透過多種路徑達到抑癌效果，包括抑制致癌基因(Oncogene)、血管生成和促生長因子；然而，在現今癌症研究顯示，Metformin在癌症治療的效應，包括與其他抗癌藥物合併治療的影響甚至在癌細胞中的作用機轉還有很多部分是未知的。此外，Emodin是一種在中草藥中常見的成分，具有抗發炎，抗氧化和肝保護功能，其抗癌活性在近期也有廣泛的研究，且Emodin可抑制多種癌細胞生長和與促使其凋亡，並且通過誘導AR降解而抑制攝護腺癌細胞生長。因此，本篇論文想探討Metformin與Emodin兩者對AR有不同抑制作用的藥物，在合併處理下，對於抑制AR蛋白穩定性、AR mRNA表現及協同抑制攝護腺癌細胞株生長的情形。研究結果發現，在Metformin 8 mM與Emodin 10 µM合併處理組發現抑制AR蛋白量的能力明顯增強，在量化統計分析也有顯著差異；而以Emodin 10µM作為對照組，在Metformin與Emodin合併處理發現細胞數量隨著Metformin的增加而隨之下降，在Metformin 4、8 mM兩組具有顯著差異，在Metformin 8 mM與Emodin 10 µM合併處理組有最高CI (Combination index)值2.537，代表在其濃度處理下具有協同作用。在AR降解方面，Metformin 8 mM與Emodin 10 µM處理下會使LNCaP細胞株中的AR蛋白質降解速度上升。在基因表現方面，Metformin 2、4、8 mM搭配Emodin 10 µM處理下與對照組比較經統計分析有顯著差異，得知Emodin對於Metformin抑制AR mRNA的表現具有加強作用。依上述結果得知，在兩者藥物合併處理下，對於LNCaP生長顯著抑制且具有協同作用；合併處理可以減少Metformin與Emodin的劑量和副作用。期許透過本篇研究的發現，能夠對於未來攝護腺癌治療方面有所貢獻。

Prostate cancer has a high incidence and mortality rate in the world. At present, many studies have shown that the occurrence and deterioration of prostate cancer are closely related to androgen receptor (AR). It is known that AR increases the growth, recurrence and metastasis of androgen-independent prostate cancer. Therefore, inhibition of AR can be one of the ways to treat prostate cancer. Our group has published that AR Ser81 site phosphorylation is important for the stability of AR protein. In addition, Metformin which is a clinical drug for diabetes patients has the anti-cancer ability including inhibitions of oncogenic ability, angiogenesis, and growth factor effects. Interestingly, Metformin was found to inhibit AR-related signaling by decreasing AR expression. On the other hand, Emodin, a compound in Chinese herbal medicine, has anti-inflammatory, anti-oxidant, and liver protective functions. Its anti-cancer activity has also recently been extensively studied such as the inhibitions of cancer growth and induction of apoptosis. Besides, Emodin can inhibit prostate cancer cell growth by degrading AR protein. Therefore, the aim of this study is to explore the effects of the combination treatment of Metformin and Emodin on prostate cancer cell line, LNCaP, and investigate whether AR protein degradation is the target of the treatment. After identifying the individual efficiencies of Metformin and emodin to AR protein decrease in LNCaP cells, we found the further decrease of AR protein level with the combination treatment and the AR decline was also identified as proteasome-dependent degradation. In this study, it was found that the combination of Metformin 8 mM and Emodin 10 µM showed that the ability to significantly inhibit AR protein level, and there were significant differences by quantitative statistical analysis. Comparing the combination treatment group Metformin 8mM and Emodin 10 µM to control group Emodin 10 µM, that the cell number was decreased following by increasing concentration of Metformin. In addition, Metformin 4 mM and 8 mM had significant differences. The Metformin 8 mM and Emodin 10 µM combined treatment group had the highest Combination Index value (CI=2.537), representing a synergistic effect under this combination. In terms of AR degradation, combined treatment with Metformin 8 mM and Emodin 10 µM could accelerate AR protein degradation in LNCaP cell line. In terms of gene expression, there was also a statistical difference between combination treatment and with the control group. It is known that Metformin can enhance the effect of Emodin in inhibiting AR mRNA. At last, LNCaP cell growth was significantly inhibited by combination treatment of Metformin and Emodin in a synergistic manner. We hope these findings might contribute to the future treatment for prostate cancer by using the strategy of combination treatment of Metformin and Emodin.

目錄
第一章、前言 1
一、背景 1
(一)、攝護腺與攝護腺癌介紹 1
(二)、雄性激素與雄性激素受體 3
(三)、Metformin介紹 4
(四)、Emodin介紹 5
二、研究動機與目的 7
第二章、材料與方法 8
一、細胞培養(Cell culture) 8
二、蛋白質萃取以及定量分析 9
(一)、蛋白質萃取(Protein Extraction) 9
(二)、蛋白質濃度測定(Bradford Assay) 9
三、細胞蛋白核質分離(Nuclear/Cytosol Fractionation) 10
四、西方墨點法(Western blotting) 11
(一)、聚丙烯醯胺膠體電泳法(SDS-Polyacrylamide Gel Electrophoresis, SDS-PAGE) 11
(二)、蛋白質轉漬法(Immunoblotting) 12
五、mRNA表現分析 14
(一)、RNA萃取(RNA purification) 14
(二)、反轉錄聚合酶連鎖反應(RT-PCR, reverse transcription polymerase chain reaction) 14
(三)、定量即時聚合酶連鎖反應(Quantitative RT-PCR) 15
六、細胞生長分析(Cell Counting) 16
七、量化及統計分析(Quantify and statistics) 16
八、實驗藥物(Drugs) 17
第三章、實驗結果 18
一、攝護腺癌細胞株LNCaP生長速率 18
二、Metformin抑制AR蛋白質表現 19
三、Emodin抑制AR蛋白質表現 19
四、Metformin與Emodin合併處理抑制AR蛋白質表現 20
五、Metformin與Emodin合併處理抑制細胞質與細胞核的AR蛋白質表現 21
六、Metformin與Emodin對於攝護腺癌細胞株LNCaP生長具有協同抑制作用 21
七、Metformin與Emodin增加AR蛋白質的降解速度 22
八、Metformin與Emodin增加AR蛋白質的降解 23
九、AR mRNA的表現量受到Metformin與Emodin抑制 24
第四章、結果與討論 26
一、Metformin對AR蛋白質之影響 26
二、 Emodin對AR蛋白質之影響 27
三、 Metformin與Emodin合併處理對LNCaP之影響 28
四、 更深入探討Metformin與Emodin對於細胞生長協同抑制作用的機制 29
第五章、結論 31
第六章、參考文獻 32
第七章、實驗結果圖 38 

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