指導教授：陳彥榮臺灣大學：生化科技學系涂琬瑛Twu, Woan-IngWoan-IngTwu2014-11-262018-07-062014-11-262018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261607當肝臟受到長期損傷會產生纖維化的現象，而肝纖維化往往會更進一步惡化成肝硬化甚至是肝癌。慢性肝病及肝纖維化在台灣及美國都是前十大主要死因之一，但目前並沒有有效的治療方式。 在肝纖維化時，胞外基質的過度累積會影響氧氣擴散造成組織內產生缺氧的情形，而 hypoxia inducible factor 1 α (HIF1α) 是體內主要調控缺氧反應的分子。在本篇研究中，我發現在纖維化的肝臟當中， HIF1α 的 mRNA 和蛋白質表現量都會上升。這些會表現的細胞同時也會表現 epithelial adhesion molecule (EpCAM) 、 Albumin (Alb) 和 Cytokeratin 19 (CK19) ，顯示這些細胞很有可能是肝臟前驅細胞。同時我也發現 β-catenin 在纖維化的肝臟中有進核的情形，表示典型 Wnt 訊息傳遞路徑是有被活化的。這些實驗結果顯示肝臟前趨細胞在纖維化的肝臟當中是處於低氧的狀態，而且 HIF1α 可能會透過 Wnt 訊息傳遞路徑調控肝臟前趨細胞。 在細胞實驗方面，我在正常氧濃度及低氧環境下培養肝癌細胞株 Huh7 ，並分析在動物實驗中有提到的基因及蛋白質的表現量。實驗結果顯示在低氧環境下，肝臟前驅細胞的標記分子 EpCAM 和 CK19 ，以及 Wnt/β-catenin 訊息傳遞路徑的下游基因 Axin2 和 CyclinD1 的 mRNA 表現量都有上升的情形； EpCAM 和 CK19 的蛋白質表現量也有上升，也可以看到 β-catenin 有進核的情形。這些研究結果顯示在動物實驗中觀察到的肝臟前趨細胞標記以及 Wnt/β-catenin 訊息傳遞路徑活化的情形很有可能是被低氧狀態所誘導的。Hepatic fibrosis is the response of liver encountering chronic injury, which would often progress to liver cirrhosis or even hepatocellular carcinoma (HCC). Chronic liver disease and liver fibrosis were in the list of top tenth cause of death in both Taiwan and the US, but no effective drug was developed for the treatment of fibrosis. The accumulation of extracellular matrix (ECM) during liver fibrosis would lead to hypoxia due to the blockage of oxygen diffusion, and hypoxia inducible factor 1 α (HIF1α) is the main modulator of hypoxic response. Here in my research, I have detected upregulated expression of HIF1α, in mRNA as well as in protein level in fibrotic liver. The cells expressing HIF1α also expressed epithelial cell adhesion molecule (EpCAM), Albumin (Alb) and cytokeratin 19 (CK19), suggesting that these HIF1α + cells were hepatic progenitor cell (HPC)-like. I also observed translocated β-catenin in fibrotic liver, indicating the activation of canonical Wnt signal. These data proved that HPCs were localized in hypoxic area during liver fibrosis, and HIF1α may interact with Wnt/β-catenin signaling to regulate HPC. As for the in vitro experiment, I cultured HCC cell line Huh7 under normoxic and hypoxic conditions to compare the expression levels of the genes mentioned in the in vivo experiment and found out that the mRNA expression level of HPC markers, EpCAM and CK19, and Wnt/β-catenin downstream targets, Axin2 and CyclinD1, were all elevated. Increased EpCAM and CK19 protein expression, in addition to β-catenin translocation could also be observed in hypoxic cultured Huh7. These results suggested that the increased expression of HPC marker and Wnt/β-catenin signaling pathway observed in vivo could be regulated by hypoxic condition.口試委員審定書 i Acknowledgement ii Abstract iii 中文摘要 v Table of Contents vi Chapter 1. Literature Review 1 1.1. Liver fibrosis and disease progression 1 1.2. Hepatic Progenitor cell (HPC) and liver disease 3 1.3. Hypoxia, hypoxia-inducible factors (HIFs) and liver disease 5 1.4. Epithelial cell adhesion molecule (EpCAM) and liver disease 7 1.5. Wnt signaling pathway and liver disease 8 Chapter 2. Aim and Experimental Flowchart 11 2.1 Motivation and Aim 11 2.2 Flowchart 11 Chapter 3. Methodology 13 3.1. Animal and CCl4 model 13 3.2. Whole liver RNA extraction 13 3.3. Hematoxylin and eosin (H&E) staining 14 3.4. Masson’s Trichrome staining 14 3.5. Liver immunofluorescence staining 14 3.6. Laser microdissection (LMD) and RNA isolation 15 3.7. Cell culture 16 3.8. Cellular RNA extraction 16 3.9. Cellular protein extraction 17 3.10. Quantitative reverse transcription PCR (qRTPCR) 17 3.11. Western blotting 18 3.12. Flow cytometry 19 3.13. Cell immunofluorescence staining 20 3.14. Image processing 21 3.15. Statistics 21 Chapter 4. Results 22 4.1 CCl4 induced fibrosis in C57BL/6 mice liver 22 4.2 Upregulated HIF1α in fibrotic liver 22 4.3 Identification of HIF1α positive cells as HPC-like cells 23 4.4 Upregulated Wnt in HPC-like cell 24 4.5 Upregulated EpCAM, Wnt and CK19 under hypoxic cultured Huh7 25 Chapter 5. Discussion and Conclusion 26 Chapter 6. Figures and Tables 29 Table 1. List of antibodies 29 Table 2. List of qRTPCR primers 31 Figure 1. H&E staining of mice liver 33 Figure 2. Masson’s trichrome staining of mice liver 34 Figure 3. Fibrotic marker mRNA expression level in whole mice liver 35 Figure 4. Immunofluorescence staining of HIF1α in mice liver 36 Figure 5. mRNA expression level of HIF1α in whole mice liver 37 Figure 6. Fibrotic markers and HIF1α mRNA expression level in LMD liver 38 Figure 7. Immunofluorescence staining of HIF1α and HPC markers 39 Figure 8. mRNA expression level of HPC markers in LMD liver 41 Figure 9. Immunofluorescence staining of Wnt/β-catenin signaling activation 42 Figure 10. mRNA expression level of Wnt downstream targets in LMD liver 44 Figure 11. Confirmation of Huh7 hypoxic culture by HIF1α and HIF2α proteins 45 Figure 12. Confirmation of Huh7 hypoxic culture by HIFs downstream targets 46 Figure 13. mRNA expression level of HPC markers in Huh7 47 Figure 14. mRNA expression level of Wnt downstream target genes in Huh7 48 Figure 15. EpCAM protein expression in Huh7 49 Figure 16. CK19 protein expression in Huh7 50 Figure 17. Immunofluorescence staining of β-catenin in Huh7 51 Figure 18. Possible mechanisms 52 Reference 53 Appendix 62 List of Abbreviations 621890210 bytesapplication/pdf論文公開時間：2019/07/29論文使用權限：同意無償授權肝纖維化低氧肝臟前驅細胞EpCAMHIFWnt/β-cateninthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261607/1/ntu-103-R01b22009-1.pdf