B型肝炎病毒X蛋白活化肝癌中Wnt/β-catenin訊息傳遞路徑之研究

Activation of Wnt/β-catenin pathway in HBx induced liver cancers

陳怡孜(Yi-Tzu Chen)

指導教授 ： 葉秀慧

國立臺灣大學/醫學院/微生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10603

摘要

Wnt/β-catenin的訊息傳遞在超過50∼70%的肝癌中有不正常的高量表現，其中有部份是因為β-catenin產生活化性突變而導致。文獻也指出，在C型肝炎相關肝癌中β-catenin產生突變的機率比B型肝炎相關肝癌還要高，這暗示著可能有一個特殊的機制，可使B型肝炎相關肝癌不藉由β-catenin的突變就可以啟動Wnt/β-catenin的訊息傳遞。於是我們有興趣藉由肝臟特定性β-catenin剔除與HBx轉殖基因 (HBx;Alb-Cre;Ctnnb1flx/flx mice)的小鼠模式來研究此一議題。
本實驗室先前的研究指出，肝臟特定性β-catenin剔除小鼠(Alb-Cre;Ctnnb1flx/flx mice)，長期追蹤後(大於16個月)，會自然產生肝腫瘤。而在肝臟特定性β-catenin剔除與HBx轉殖基因的小鼠模式中，因為HBx蛋白的存在，會使腫瘤生成的時間提早，從第七到第八個月開始就會產生肝腫瘤。
我們首先釐清Wnt/β-catenin 訊息傳遞路徑在本實驗室小鼠模式所產生的肝腫瘤中是否活化。結果顯示，在肝臟特定性β-catenin剔除小鼠的肝腫瘤會藉由β-catenin的突變來活化Wnt/β-catenin 訊息傳遞路徑；在肝臟特定性β-catenin剔除與HBx轉殖基因小鼠的肝腫瘤中也發現有部分的Wnt/β-catenin 訊息傳遞路徑是被活化的，但這些腫瘤中的β-catenin都沒有發生突變。為了進一步探討是什麼樣的機制去活化這群腫瘤中的Wnt/β-catenin 訊息傳遞路徑，我們將目標放在發炎反應所造成的微環境中。我們發現在肝臟特定性β-catenin剔除與HBx轉殖基因小鼠的肝臟，在第四週開始會發生細胞衰老(senescence)現象，使β-catenin被剔除的肝細胞死亡，引起發炎反應。和發炎反應相關的因子中，補體C1q最近被證實可參與活化Wnt/β-catenin 訊息傳遞路徑，而在肝臟特定性β-catenin剔除與HBx轉殖基因小鼠發炎的肝臟微環境中也觀察到C1q的表現量有增加，這可能與刺激肝臟前驅細胞活化增殖與肝癌的形成有關。在免疫組織化學染色的結果發現，C1q與分泌C1q的巨噬細胞會表現在鄰近肝臟前驅細胞與肝腫瘤的附近，顯示著C1q極有可能參與調控這些細胞的Wnt/β-catenin 訊息傳遞路徑。
最後我們試著去驗證C1q相關的機制是否也存在於人類肝癌中。結果發現在85%的人類肝癌中，其非腫瘤部分比起腫瘤區的C1q表現量有增加的趨勢，其中有部分檢體的Wnt/β-catenin訊息傳遞路徑下游基因Axin2的表現也有增加。這初步的結果支持著在發炎微環境中的C1q，可能是與活化人類肝癌中Wnt/β-catenin 訊息傳遞路徑相關，更詳細的機制則有待進一步的研究來驗證。

關鍵字

Wnt/β-catenin訊息傳遞路徑 ； B型肝炎 ； X蛋白 ； 肝癌

並列摘要

It has been well documented that the Wnt/β-catenin pathway is activated in 50-70% of human hepatocellular carcinoma(HCC), some of which is attributed by the somatic mutations. It was noted that the somatic mutations occurs less frequently in HBV-related HCC than the HCV- related HCC, suggesting a possibility that some specific mechanism might contribute to the activation of Wnt/β-catenin pathway in HBV- related HCC. The current study proposed to address this possibility by using the HBx induced HCC mouse model, with conditional β-catenin knockout mice in crossing with HBx transgenic mice (HBx;Alb-Cre;Ctnnb1flx/flx mice). In our previous study, liver cancers occur spontaneously in the β-catenin knockout mice (Alb-Cre;Ctnnb1flx/flx mice) during long term follow-up (at the age >16 months old). The presence of HBx can accelerate the carcinogenesis, with the liver tumors occurred earlier at the age 7~8 months old.
We have first tried to examine if the Wnt/β-catenin pathway is activated in the liver tumors of this animal model. The results showed that most of the liver tumors in β-catenin knockout mice are attributed by the somatic activating mutations. However, although the Wnt/β-catenin pathway is also activated in a subgroup of β-catenin knockout with HBx transgenic mice, none of which are attributed by activating mutations. Aiming to delineate the mechanisms for activation of Wnt pathway in this subgroup of HCC, we focused on the candidate factors in the inflammatory microenvironment for this activation event. We found that this inflammatory microenvironment is predisposed by senescence of the β-catenin deficient hepatocytes, starting from 4 weeks of age. A novel factor of C1q has recently been identified with ability to activate Wnt pathway, which intriguingly is significantly elevated in the inflammatory niche of thisβ-catenin knockout with HBx transgenic mice and is well correlated with the expansion of the HPCs and carcinogenesis. The results from extensive IHC staining further revealed the C1q positive cells and the C1q secreting macrophages in close proximity of the HPCs and liver cancers, supporting the possible involvement of this complement factor in regulating Wnt pathway in these cells. Finally, we have also tried to examine if this mechanism could also occur in human HCC. The results showed that the expression of C1q is elevated in the non-tumorous liver tissues adjacent to HCC in 85% of HCC patients, some of which correlate with the increase of Axin, a target gene of Wnt pathway, in HCC. The results thus preliminarily supported the C1q as one putative factor in the inflammatory microenvironment associated with activating the Wnt pathway in human HCC, which awaits further validation studies.

並列關鍵字

Wnt/β-catenin pathway ； HBx ； liver cancers

參考文獻

參考文獻

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