臺灣博碩士論文加值系統

胰管腺癌和膽管癌是兩種最致命的癌症，五年存活率皆低於10%。近年來，有許多可能參與在胰管腺癌與膽管癌形成的基因逐漸被發現。利用癌症基因組圖譜資料庫進行初步分析，我們發現DPH1表現量較高的胰管腺癌患者有較好的存活率，但是在膽管癌患者中，DPH1表現量較高的病患則是有較差的存活率，然而我們目前仍然不清楚DPH1在胰管腺癌與膽管癌中所扮演的角色。為了研究DPH1在胰管腺癌與膽管癌起始 (initiation) 中所扮演的角色，我們利用誘導性Keratin 8 (K8)-Cre系統建立了上皮特異性 (epithelial-specific) Dph1剔除小鼠模型。我們發現在小鼠胰管細胞與膽管細胞單獨剔除Dph1不足以促使癌症發生，我們便假設Dph1可能不會參與在癌症起始，而是參與在癌症形成的過程中。因此，我們便轉而在K8-CreERT2; KrasG12D/+; Ptenfx/+的背景下探討Dph1在胰管腺癌與膽管癌形成過程中所扮演的角色，其中KrasG12D/+; Ptenfx/+在近年來的研究中已知能夠造成胰管腺癌與膽管癌的形成。我們發現Dph1缺失對於Kras突變與Pten異型合子缺失所造成的胰管腺癌沒有太大的影響，然而Dph1缺失能夠減輕由Kras突變與Pten異型合子缺失所造成膽管細胞增生 (hyperplasia)，顯示KRAS活化與PTEN缺失的膽管癌形成需要Dph1的存在。為了更進一步研究其背後的機制，我們建立了帶有Kras/Pten/Dph1複合基因突變 (compound mutant) 且能夠重現胰管腺癌與膽管癌組織病理特徵的胰管類器官與膽管類器官。儘管Dph1缺失對於KRAS/RAF/MEK/ERK訊息傳遞沒有明顯的影響，但是卻能夠降低胰管類器官與膽管類器官的增生以及整體mRNA轉譯效率。總結來說，我的研究表明在致癌性Kras活化與Pten異型合子缺失的背景下，Dph1缺失透過降低mRNA轉譯效率進而抑制胰管腺癌與膽管癌增生。

Pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CC) are two of the deadliest cancers. The 5-year survival of both PDAC and CC are less than 10%. Recently, many genes have been discovered that may be involved in PDAC and CC formation. Using The Cancer Genome Atlas (TCGA) database for preliminary analysis, we found that PDAC patients with high DPH1 levels have better survival. In contrast, CC patients with high DPH1 levels have lower survival. However, the roles of DPH1 in PDAC and CC remain unclear. To investigate the roles of DPH1 in PDAC and CC initiation, we generate epithelial-specific Dph1 conditional knockout mice using inducible keratin 8 (K8)-Cre system. We found that loss of Dph1 alone in pancreatic ductal cells and cholangiocytes is insufficient to drive tumor formation. We hypothesized that Dph1 may not play a role in tumor initiation but tumor progression. Hence, we investigate the role of Dph1 in PDAC and CC progression in the context of K8-CreERT2; KrasG12D/+; Ptenfx/+, which is known to develop PDAC and CC in recent studies. We found that loss of Dph1 confers comparable phenotype in K8-CreERT2; KrasG12D/+; Ptenfx/+ PDAC. On the other hand, we found that loss of Dph1 mitigates K8-CreERT2; KrasG12D/+; Ptenfx/+ cholangiocyte hyperplasia, suggesting that Dph1 is required for KRAS-activated and PTEN-defective CC progression. To further investigate the underlying mechanisms, we established Kras/Pten/Dph1 compound mutant pancreatic ductal organoid and cholangiocyte organoid, which successfully recapitulates PDAC morphology and CC morphology. Although loss of Dph1 confers no overt effect on KRAS/RAF/MEK/ERK signaling, we found that Dph1 deficiency reduces the proliferation and mRNA translation efficiency of both pancreatic ductal organoid and cholangiocyte organoid. In summary, the findings of my thesis suggest that Dph1 deficiency suppresses PDAC and CC proliferation through reducing mRNA translation efficiency in the context of heterozygous Pten loss and oncogenic Kras activation.

目錄
中文摘要 iv
英文摘要 v
壹、緒論 1
一、胰管腺癌 1
二、膽管癌 2
三、DPH1的生理功能與相關癌症 2
四、KRAS在胰管腺癌與膽管癌中的角色 3
五、PTEN在胰管腺癌與膽管癌中的角色 4
六、類器官培養系統 5
貳、研究目的 7
參、實驗材料與方法 8
一、實驗小鼠 8
二、基因體DNA萃取 9
三、聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 確認基因型 9
四、Tamoxifen (10 mg/ml) 配製與施打 10
五、組織固定、脫水、石蠟包埋與切片 10
六、類器官 (Organoid) 建立與培養 10
七、以4-OHT (4-hydroxytamoxifen) 處理類器官的方式 13
八、類器官固定、脫水、石蠟包埋與切片 13
九、蘇木精與伊紅染色 (Hematoxylin & Eosin staining, H&E staining) 13
十、免疫螢光染色 (Immunofluorescence staining, IF staining) 14
十一、細胞增殖試驗 (Cell proliferation assay) 14
十二、嘌呤黴素標記 (Puromycin labeling) 15
十三、類器官蛋白質萃取與西方墨點法 (Western Blot, WB) 15
十四、抗體 (Antibody) 16
十五、生物資訊與統計分析 17
肆、實驗結果 18
一、Dph1剔除對於胰管腺癌與膽管癌生成的影響 18
二、Dph1在Kras突變下對於胰管腺癌與膽管癌進程的影響 18
三、Dph1在Kras突變與Pten缺失下對於胰管腺癌與膽管癌進程的影響 19
四、利用類器官系統探討Dph1影響胰管腺癌與膽管癌進程的機制 21
伍、討論 25
一、Dph1在胰管腺癌與膽管癌中所扮演的角色 25
二、Dph1在胰管腺癌中的角色於小鼠模型與類器官系統有所不同 26
三、Dph1異型合子缺失之表現型 (phenotype) 理論上應該要類似Dph1野生型之表現型 26
四、控制組在以類器官系統為模型的研究中無法重現真實體內情況 27
五、結論 28
陸、參考文獻 29
柒、圖表 33
圖一、單獨剔除Dph1不足以驅動胰管腺癌與膽管癌的形成 34
圖二、Dph1缺失對於帶有Kras突變的胰管與膽管沒有明顯的影響 36
圖三、Dph1不影響由Kras突變與Pten異型合子缺失所造成的胰管腺癌進程 38
圖四、Dph1缺失似乎有助於胰管腺癌細胞增生 39
圖五、Dph1缺失不影響KRAS/RAF/MEK/ERK訊息傳遞 40
圖六、Dph1剔除能夠抑制由Kras突變與Pten異型合子缺失所引起的肝臟外膽管肥大以及肝臟內膽管增生 41
圖七、Dph1剔除能夠抑制由Kras突變與Pten異型合子缺失所引起的肝臟內膽管增生 42
圖八、Dph1剔除抑制了KRAS/RAF/MEK/ERK訊息傳遞 44
圖九、4-OHT誘導成功建立能夠重現小鼠胰管腺癌的類器官系統 46
圖十、Dph1缺失能夠抑制帶有Kras突變與Pten異型合子缺失的胰管類器官增生 48
圖十一、Dph1缺失不影響胰管類器官之KRAS/RAF/MEK/ERK訊息傳遞 50
圖十二、Dph1缺失造成帶有Kras突變與Pten異型合子缺失的胰管類器官整體mRNA轉譯效率下降 52
圖十三、4-OHT誘導成功建立能夠重現小鼠膽管癌的類器官系統 54
圖十四、Dph1缺失能夠抑制帶有Kras突變與Pten異型合子缺失的膽管類器官增生 56
圖十五、Dph1缺失不影響膽管類器官之KRAS/RAF/MEK/ERK訊息傳遞 58
圖十六、Dph1缺失造成帶有Kras突變與Pten異型合子缺失的膽管類器官整體mRNA轉譯效率下降 60
捌、附錄 61
附錄圖一、DPH1表現量似乎和胰管腺癌病患與膽管癌病患存活率有關 61

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