臺灣博碩士論文加值系統

隨著社會節奏加快，忙碌的現代人生活習慣開始跟著改變，不良的生活習慣，慢慢的侵犯人體。目前國人女性罹患子宮體癌比例已超越子宮頸癌以及卵巢癌成為女性生殖道癌症第一名，而罹患歲數已有年輕化的趨勢。大自然植物界為豐富的資源庫，植物中蘊藏許多可利用的生物活性並且具有相當潛力發展成於醫療藥物。本篇論文使用民間中草藥廣藿香以及其有效化合物廣藿香醇對於第一型子宮內膜癌細胞株之抗腫瘤作用，藉由探討細胞增殖、細胞週期及細胞凋亡等相關機制，發現以水萃取之廣藿香 (Pogostemon cablin；PC) 化合物可抑制子宮內膜癌細胞株增殖並且介導粒線體去極化使其走向細胞凋亡。廣藿香相關化合物，其中以廣藿香醇 (PatchouliAlcohol; PA) 對於子宮內膜癌細胞株最具有抗增殖作用，以引發粒線體去極化而活化 caspase 9 以及 caspase 3 方式造成細胞凋亡現象，並也造成細胞週期G1期停滯，先以即時聚合酶鏈式反應 (Real time polymerase chain reaction; RealTime-PCR) 檢測基因表達，G1 期調控基因 CDK4、Cyclin E 和 Cyclin D 表達下降，而位於細胞週期上游條控基因 p27, p21 以及 p53 表達上升，再以西方墨點法檢測蛋白質表達，調控 G1 期之蛋白 CDK4、Cyclin D 以及 Cyclin E/CDK2 之間結合力表達皆下降。但引發細胞週期上游調控蛋白 p53、p27 則未改變，藉由Human Cell Cycle PCR Array具有多樣性的基因進行分析，針對其中呈現上調之RAD9 homolog A (S. pombe) (RAD9) 基因進行探討，發現RAD9蛋白表達上升並且與RAD9B之間結合力上升，進而調控下游蛋白CHK2以及CDC25A的磷酸化造成 G1 期停滯。

關鍵字:廣藿香; 廣藿香醇; 細胞凋亡; 細胞週期G1期停滯; RAD9

The quickening pace of modern life is promoting unhealthy living habits that gradually take their toll on the human body. Uterine cancer has surpassed cervical cancer and ovarian cancer to become the most common forms of female reproductive system cancer in Taiwan, Furthermore, the average age at uterine cancer occurs is showing a downward trend. A wide range of bioactive resources with the potential to become medical drugs exist in the plant kingdom. This study examined the anti-tumor effects of the Chinese medicinal herb Pogostemon cablin (PC) and its active compound Patchouli Alcohol (PA) on Type I endometrial cancer cells. In our examination, it was discovered that the water extract of PC can inhibit the proliferation of endometrial cancer cells and mediate mitochondrial depolarization, thereby inducing apoptosis. Among the compounds of PC, PA was shown to be the most effective in inhibiting the proliferation of endometrial cancer cells and promoting cell apoptosis by inducing mitochondrial depolarization and activating caspase 9 as well as caspase 3, resulting in G1 cell-cycle arrest. We employed real-time polymerase chain reaction to detect gene expression as well as the Human Cell Cycle PCR Array to investigate upregulated RAD9 genes. The protein genes CDK4, Cyclin E, and Cyclin D, which regulate the G1 cell cycle, were shown to be downregulated, whereas upstream regulatory genes p27, p21, and p53 were upregulated. Western blot analysis revealed downregulation in the expression of CDK4 and Cyclin D and the binding forces in Cyclin E/CDK2. However, no changes were observed in the expression of upstream regulatory genes p53 or p27. The expression of the RAD9 protein increased, and the binding force with its paralog RAD9B increased, which regulate the phosphorylation of downstream protein CHK2 and phosphatase CDC25A, thereby resulting in G1 cell-cycle arrest.

Keywords:Pogostemon cablin; Patchouli Alcohol; Cell cycle G1 phase arrest; RAD9

目錄
頁次
英文縮寫 III
中文摘要 IV
英文摘要 VI
壹、導論 1
一、子宮內膜癌 1
二、子宮體癌類型 2
三、廣藿香 3
四、細胞週期調控 5
五、細胞凋亡 7
六、人類 RAD9 蛋白 8
貳、研究材料與方法 9
一、細胞培養 9
二、藥物配置 9
三、細胞活性測試 9
四、細胞計數方法 10
五、細胞凋亡分析 11
六、細胞週期分析 12
七、Caspase 3活性分析 14
八、粒線體膜電位分析 （Mitochondrial Membrane Potential; ΔΨm） 15
九、西方墨點法 (Western Blot) 15
十、免疫共沉澱法 (Co-Immunoprecipitation) 19
十一、總細胞RNA的純化分離 19
十二、反轉錄反應 (Reverse transcription polymerase chain reaction) 20
十三、即時聚合酶鏈式反應 (Real time polymerase chain reaction) 21
十四、Human Cell Cycle RT² Profiler™ PCR Array分析 21
十五、統計分析 22
參、研究構想 23
肆、結果 24
一、廣藿香水萃取物對子宮內膜癌細胞株細胞增殖影響 24
二、廣藿香水萃取物對子宮內膜癌細胞株細胞凋亡影響 24
三、廣藿香水萃取物對子宮內膜癌細胞株細胞週期影響 24
四、檢測細胞凋亡相關蛋白質表達 25
五、檢測粒線體膜電位的變化 25
六、分析廣藿香中有效化合物對子宮內膜癌細胞株細胞增殖的影響 26
七、廣藿香醇對子宮內膜癌細胞株細胞增殖型態的影響 26
八、廣藿香醇對子宮內膜癌細胞株細胞增殖的影響 27
九、廣藿香醇對子宮內膜癌細胞株細胞凋亡的影響 27
十、廣藿香醇對子宮內膜癌細胞株細胞週期影響 27
十一、廣藿香醇對子宮內膜癌細胞株細胞凋亡相關蛋白質表達 28
十二、檢測廣藿香醇對子宮內膜癌細胞株之粒線體膜電位變化 28
十三、廣藿香醇對子宮內膜癌細胞株之細胞週期與凋亡相關基因影響 29
十四、Human Cell Cycle PCR Array分析 29
十五、廣藿香醇對子宮內膜癌細胞株細胞內調控 G1 期蛋白質表達 29
十六、RAD9 蛋白調控子宮內膜癌細胞株細胞細胞週期 G1 期的影響 30
伍、討論 31
陸、結論 38
柒、文獻 39
附表 52
附錄 55
附圖 57

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