臺灣博碩士論文加值系統

肝癌的致死率在目前所有癌症中仍名列前茅，其中最常看到的肝癌種類為肝細胞癌(Hepatocellular carcinoma, HCC)。在臨床和流行病學研究中顯示肝細胞癌的成因中最常見的則是肝臟慢性發炎而轉變成肝細胞癌的狀況。在細胞的發炎反應中類鐸受器扮演了一個重要的角色。TLR4為已經研究相當透徹的類鐸受器成員。可以活化TLR4的促進劑中最常被提出的為LPS。在我們先前的實驗中看到以肝癌細胞長時間培養於含有LPS的環境中持續活化TLR4可以使細胞中TLR4表現量增加，以及因為let-7的功能被抑制，所以使得肝癌細胞容易轉變成具有癌症前驅性特性的細胞。然而因為在臨床上較少有因為LPS刺激而促使肝癌發展的研究被提出，所以我們便假設是否在肝臟的環境中含有可以促進肝癌細胞生長的物質。在此篇研究中我們將肝癌細胞長時間培養於收集肝癌細胞Huh7培養24小時的培養液Huh7 CM，由結果可以觀察到在長時間處理Huh7 CM後同樣可以使肝癌細胞中的TLR4表現量增加。另外，在此篇研究中為了證實這些轉變為具有癌症前驅性特性的肝癌細胞是否可以影響生物體內腫瘤的發展，所以我們將經過長時間處理LPS且TLR4表現量上升的肝癌細胞注射到實驗小鼠體內觀察其對於腫瘤生長的影響。結果顯示TLR4表現量增加的肝癌細胞確實可以促進腫瘤的生長與惡化。除此之外我們也想要徹底的了解Huh7 CM中是哪種物質可以影響TLR4的活性，在先前的實驗中我們篩選並分析出胱抑素C是最有可能影響肝癌細胞生長的內源性分子。所以在我們此篇研究中另一個主軸便是想要釐清胱抑素C是否可以如同LPS一樣可以活化TLR4並且促進肝癌細胞的生長。我們藉由將肝癌細胞Huh7直接處理胱抑素C並觀察細胞中TLR4的下游訊息傳遞活化情形，由結果觀察到胱抑素C確實可以促進TLR4的活化。另外我們也藉由colony formation assay來觀察胱抑素C對於肝癌細胞的生長影響，實驗結果顯示出，當Huh7細胞處理胱抑素C後可以使colony生成的數目增加。最後我們更想要連結胱抑素C促進肝癌細胞的生長作用是透過活化TLR4的下游訊息傳遞來進行的，因此我們將Huh7細胞中的TLR4 knockdown並處理胱抑素C，由實驗結果我們可以看到處理胱抑素C後TLR4 knockdown的Huh7細胞colony數目增加的現象被抑制了。所以總結實驗結果我們確定了肝癌Huh7可以分泌胱抑素C到細胞外環境中，透過活化TLR4的下游訊息傳遞來促進肝癌細胞的生長。

Hepatocellular carcinoma is the most common type of primary liver cancer in adults, which is usually accompany with high lethality. Shariff, M.I. et al. indicate that chronic inflammation of liver is the major risk factor for HCC development, and toll-like receptors, play important roles in the inflammatory response. In our preliminary data, sustained TLR4 activation by LPS increased TLR4 expression and stemness property in HCC cell lines. However, there are rare record indicating the association of LPS with HCC. According to these results, we want to further confirm the stemness property in HCC cell lines in vivo, and we hypothesize that endogenous molecules induced by long-term LPS exposure secreted from liver cells, instead of LPS promote TLR4-dependent cell proliferation. In this study, PLC5 cells with LPS long-term treatment were subcutaneous injected into SCID mice, and the results show that the growth and progression are increased in the line with LPS long-term treatment. Meanwhile, the TLR4 expression is increased in HCC cells with Huh7 CM long-term treatment. In previous studies, Cystatin C show as the candidate of endogenous molecule that activates TLR4 pathway in Huh7 CM. Thus, we want to investigate whether Cystatin C promotes HCC cells proliferation through TLR4 activation. To demonstrate that, Huh7 cells were treated with Cystatin C recombinant protein, and the result indicates that TLR4 downstream signal pathway was activated, and the growth of HCC cells was promoted by Cystatin C treatment. Furthermore, TLR4 activation and cell proliferation was decreased when TLR4 knock-down Huh7 cells were treated with Cystatin C or Huh7 cells were treated with CM from Cystatin C silencing cells. In conclusion, we suggest that Cystatin C was secreted from Huh7 cells in extracellular environment and it promotes growth of HCC cell through activation TLR4 downstream signal pathway.

目錄
中文摘要 i
Abstract ii
中英文對照表 iii
目錄 vi
圖目錄 vii
第一章 緒論 1
1-1 腫瘤為環境與癌症之間的關係 1
1-2 慢性發炎與肝癌 2
1-3 類鐸受器在癌症中扮演的角色 3
1-4 癌症前驅細胞與TLR4 5
1-5 肝癌與TLR4 9
1-6 TLR4的促進劑 11
1-7 肝癌的發展與TLR4內源性促進劑 13
1-8 胱抑素C 15
1-9 胱抑素C與癌症 16
第二章 研究動機與目的 17
2-1 研究動機 17
2-2 研究目的 18
第三章 實驗材料與方法 19
3-1 實驗材料 19
3-2 實驗方法 23
第四章 研究結果 31
4-1 藉由Huh7 CM長時間處理肝癌細胞以模擬肝癌細胞處於腫瘤微環境中對TLR4表現量的影響 31
4-2 慢性發炎對於肝癌細胞生成腫瘤能力的影響 34
4-3 確認胱抑素C在不同癌症細胞株中的表現量 36
4-4 胱抑素C對於肝癌細胞生長的影響 40
4-5 胱抑素C對於肝癌細胞中TLR4下游訊息傳遞的活化 41
4-6 胱抑素C透過TLR4來活化TLR4下游訊息傳遞 44
4-7 胱抑素C藉由TLR4來調控肝癌細胞的生長 45
第五章 結論與討論 47
第六章 圖表 56
第七章 參考資料 66


圖目錄
圖1. 模擬PLC5處於慢性發炎的狀態下TLR4的表現變化 56
圖2. 藉由異種移植觀察經過模擬慢性發炎培養之PLC5細胞在老鼠模型中對於腫瘤生成的影響 57
圖3. 檢測Huh7 CM中胱抑素C的蛋白濃度 58
圖4. 檢測不同的癌症細胞株細胞中胱抑素C的表現量與conditioned medium中胱抑素C的濃度 59
圖5. 檢測不同的肝癌細胞株中胱抑素C的表現量及conditioned medium中胱抑素C的濃度 60
圖6. 觀察Huh7 CM中的胱抑素C含量下降時對於Huh7細胞生長情況的影響 61
圖7. 觀察存在於medium中的胱抑素C對於Huh7細胞生長情況的影響
62
圖8. 觀察處理胱抑素C後Huh7細胞內TLR4的訊息傳遞路徑活化情形
63
圖9. 觀察在進行TLR4基因敲落的Huh7細胞處理胱抑素C後，細胞內TLR4的訊息傳遞路徑活化情形 64
圖10. 觀察在Huh7細胞進行TLR4基因敲落並處理LPS、Huh7 CM及胱抑素C後對於細胞的生長影響 65

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