臺灣博碩士論文加值系統

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) 在小腸、大腸、胃、肝、髮囊和乳腺的幹細胞中都可作為細胞表面的標誌基因。近期的研究指出LGR5的表現量也可作為味蕾幹細胞的標誌物。LGR5在許多不同種類的人類惡性腫瘤幹細胞中，例如大腸癌、胃癌以及食管腺癌也會大量表現，並且LGR5的表現與腫瘤形成的過程有密不可分的關係。但LGR5在口腔癌中所扮演的角色目前並未有相關的文獻。因此，本研究計畫利用生物資訊及細胞實驗方法來探討LGR5在口腔癌的致病過程中扮演的角色及參與的功能機制。首先透過不同種類的線上資料庫評估LGR5在口腔癌組織中DNA copy number、基因表現量以及蛋白質表現量的變化，在Cancer Genome Atlas資料庫中顯示LGR5的copy number於頭頸癌中有顯著的增加。而在GSE3524的研究和Human Protein Atlas資料庫中顯示LGR5於口腔癌組織裡的表現量較高，因此RNA與蛋白質的結果一致。接著利用siRNA抑制口腔癌細胞內生性LGR5表現量，並探討其表現量下降是否影響細胞生長及下游訊號傳遞的路徑。抑制LGR5的表現量確實能夠降低細胞的生長速率，並透過流式細胞儀分析細胞週期發現多數細胞停滯在G2期。以上結果顯示LGR5於口腔癌中可能扮演致癌基因的角色，會透過調控細胞增生和細胞週期影響口腔癌的生成。

The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a well characterized surface marker for multiple type of adult stem cells, including small intestine, colon, stomach, liver, hair follicle and mammary gland. Recently, LGR5 is also found to mark the taste bud stem cells in posterior tongue. Elevated expression of LGR5 has been linked to the development of cancer stem cells in a variety of human malignancies, including colorectal cancer, gastric cancer and esophageal adenocarcinoma. However, the role of LGR5 in oral squamous cell carcinoma (OSCC) has not been reported. In this study, we aimed to explore the role of LGR5 in OSCC pathogenesis and to delineate the mechanism underlying its function in OSCC. We evaluated the change in DNA copy number, gene expression level and protein expression pattern of LGR5 in OSCC samples using multiple online databases. We established a siRNA-based technology to suppress endogenous levels of LGR5 and analyzed the effects of LGR5 depletion on cell growth, proliferation and downstream signaling in cultured OSCC cells. Using the cancer genome atlas database, we discovered that the DNA copy number of LGR5 is significantly amplified in head and neck cancer. The mRNA and protein levels of LGR5 in OSCC tissues were also found to be up-regulated based on the GSE3524 dataset and in Human Protein Atlas database. Depletion of LGR5 suppressed the proliferation and reduced the colony forming ability of cultured OSCC cells. Flow cytometry analysis revealed that depleting LGR5 in OSCC cells caused the cell cycle to arrest in G2 phase. Our findings suggest that LGR5 may function as an oncogene to promote OSCC pathogenesis by regulating the proliferation and cell cycle progression of OSCC cells.

論文指導教授推薦書……………………………………………...……..
論文口試委員會審定書…………………………………………..…..…
長庚大學博碩士紙本論文著作授權書…………………….…….….....iii
誌謝………………………………………………………………….…..iv
中文摘要………………………….……………………………….…...vi
Abstract…………………………………………………………………vii
目錄…………………………………………………………………...viii


第一章 緒論…................................................................... -1-
1-1. 癌症 ( Cancer)..................................................... -1-
1-1.1 癌症概述.......................................................... -1-
1-1.2 癌症的發展(Development of Cancer) ............................. -1-
1-2. 口腔癌 ( Oral cancer ) ................................................ -2-
1-2.1口腔癌概論.............................................................. -2-
1-2.2口腔癌之流行病學............................................................ -3-
1-2.3口腔癌之病理組織診斷.................................................... -3-
1-3. LGR5概述......................................................... -5-
1-3.1 LGR之亞型介紹............................................................ -5-
1-3.2 LGR5介紹.............................................................. -6-
1-3.3 Wnt/β-Catenin調控路徑.............................................. -7-
1-3.4 LGR5與Wnt/β-Catenin之關係.................................. -8-
1-3.5 LGR5於癌症中的角色.................................................. -9-
第二章 研究動機與目的....................................................... -10-
第三章 實驗方法.......................................................... -11-
3-1. 細胞培養 ( Cell culture ) ............................................ -11-
3-2. 萃取細胞 RNA (Cellular RNA extraction) .......................... -11-
3-3. 反轉錄聚合酶連鎖反應................................................. -12-
3-4. Q-PCR定量.......................................................... -12-
3-5. siRNA Transfection.................................................... -13-
3-6. DAPI Cell Proliferation Assay............................................ -13-
3-7. Colony Formation Assay..................................................... -14-
3-8. Cell Cycle Analysis............................................................. -14-
3-9. Annexin V/Propidium-Iodine Apoptosis Assay.................. -15-
3-10. JC-1 Staining............................................................ -15-
3-11. Protein Extraction............................................................. -16-
3-12. BCA Protein Assay...................................................... -16-
3-13. Western Blot Analysis.............................................. -17-

第四章 實驗結果....................................................... -18-
4-1. 利用線上資料庫評估LGR5於口腔癌中的表現量……..... -18-
4-2. 評估LGR5 copy number的變化及造成的突變位點…….. -18-
4-3.QPCR分析LGR5在不同口腔癌細胞株中的表現量….… -19-
4-4. OSCC細胞抑制LGR5後觀察細胞型態變化……………. -19-
4-5. OSCC細胞抑制LGR5後分析蛋白質表現量……………. -20-
4-6. OSCC細胞抑制LGR5後其細胞生長速率下降…………. -20-
4-7. OSCC細胞抑制LGR5後其形成colony能力下降………. -21-
4-8. SCC25抑制LGR5導致細胞週期停滯於G2期…………. -21-
4-9. SCC25抑制LGR5並經長時間培養後導致細胞凋亡……. -22-
4-10. OECM1細胞抑制LGR5導致細胞凋亡………….…...…. -23-
4-11. OECM1細胞抑制LGR5後細胞膜電位變化…………. -24-
4-12. OECM1細胞抑制-catenine觀察細胞生長情形……........ -25-
第五章 討論.................................................. -26-
參考文獻...................................................................... -30-
第六章 圖表附錄................................................................. -34-
圖一、LGR5於口腔鱗狀細胞癌組織中的表現量……………… -34-
圖二、LGR5在頭頸癌中的突變位點…………………….…… -35-
圖三、分析LGR5於口腔癌細胞株中的表現量………………… -36-
圖四、抑制LGR5後細胞型態的變化……………………..…… -37-
圖五、抑制LGR5後細胞中蛋白質的表現量……………..…… -39-
圖六、SCC25和OECM1抑制LGR5後細胞生長曲線….…… -40-
圖七、 SCC25和OECM1抑制LGR5後細胞構成colony的能力. -41-
圖八、SCC25抑制LGR5後其細胞週期的分佈情形…….… -42-
圖九、SCC25抑制LGR5後分析細胞凋亡程度……………… -43-
圖十、SCC25抑制LGR5後培養較長時間下的細胞週期…… -44-
圖十一、SCC25抑制LGR5後培養較長時間下細胞凋亡的程度.. -45-
圖十二、OECM1抑制LGR5後分析細胞凋亡的程度…………. -46-
圖十三、OECM1抑制LGR5後觀察細胞中粒線體膜電位的變化.-47-
圖十四、OECM1抑制-catenine後觀察細胞生長情形………… -48-

附錄............................................................................ -49-
附錄一 LGR5 結構………….…………..………………..…… -49-
附錄二LGRs之亞型分類……………………………..…… -50-
附錄三 Wnt/β-Catenin訊號傳遞路徑示意圖……..………...… -51-
附錄四 LGR5-/-小鼠舌帶短縮情形之舌頭剖面圖………….… -52-
附錄五 LGR5表現於正常小鼠之舌頭和下顎的上皮細胞..… -53-

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