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IGF-IR Signaling Participates in Cholangiocyte Proliferation during Early Stage of Intrahepatic Cholangiocarcinoma in Liver-Specific IMP2 Transgenic Zebrafish
|Authors: ||Chian-Chi Huang|
|Contributors: ||NTOU:Department of Aquaculture|
|Issue Date: ||2011-11-25T07:31:35Z
|Abstract: ||臨床研究發現IMP2在人類肝臟腫瘤包括肝癌及膽道癌均會過量表現。先前實驗室以TetOff調控系統建立肝臟專一性過量表現IMP2之基因轉殖斑馬魚品系，發現10週大斑馬魚即會產生嚴重肝臟內膽道癌(ICC)。本研究主要探討在肝臟專一性表現IMP2基因轉殖斑馬魚誘發肝臟內膽道癌癌化過程之早期階段的致病分子機制。以病理切片檢視IMP2 基因轉殖斑馬魚肝臟內膽道癌癌化過程進展，顯示從第四週肝細胞即明顯受損，到第六週肝臟內膽道細胞增生，到第10 週產生肝臟內膽道癌。從第一週到第六週處理 tetracycline 關掉轉殖IMP2 基因表現，進行石蠟切片與 H-E stain 的分析，結果顯示：關掉肝臟 IMP2過量表現可以抑制肝臟內膽道細胞增生的表現型，確認膽道細胞增生是由於 IMP2 導致的病理結果。進一步藉由免疫組織化學染色 (IHC) 利用膽道細胞專一性標誌 CK18 (cytokeratin 18) 和細胞增生標誌PCNA (Proliferation Cell Nuclear Antigen) 確定膽道細胞增生的表現型。為了瞭解IMP2在膽道增生早期階段的分子機制，進一步利用Microarray 和 Q-PCR 進行分析，發現 IGF-2b、 IGF-IRa、 IGF-IRb和 Progranulin–A (PGRN-A) 表現均在四週大斑馬魚之肝臟中被活化。結果顯示第四週是重要的階段，肝細胞過度表現 IMP2可調控下游自分泌、旁分泌生長因子如 IGF2、 PGRN-A ，進而活化IGF-IR 及PGRN-A受體訊息傳導，藉由增加磷酸化PTEN及AKT來弱化PTEN及活化AKT，促進存活/細胞增生訊息途徑，進而誘導膽道細胞增生。進一步從第三週至第六週利用 IGF-2b、 IGF-IRa、IGF-IRb及PGRN-A之vivo-Morpholinos個別腹腔注射處理 IMP2基因轉殖斑馬魚。結果顯示個別抑制 IGF-IR及IGF-IRb 可以顯著降低肝臟內膽道細胞增生的發生率自78 % 降低至50 % 及44 %。綜合以上結果顯示 IGF-IR 訊號路徑參與在IMP2誘發膽道癌早期階段在促進肝臟內膽道細胞增生確實扮演著重要的角色，可做為肝臟內膽道癌早期診斷及標靶藥物治療的標的分子。肝臟過度表現 IMP2 誘導膽道細胞病變，提供了一個新穎的動物模式以研究 ICC 疾病進展過程中肝細胞與膽道細胞間交互作用之分子機制。|
IGF2 mRNA-binding protein 2 (IGF2BP2/IMP2) was expressed in fetal liver but little in adult liver while overexpressed in the human liver tumors including hepatocellular carcinoma and cholangiocarcinoma of human. To study the molecular mechanism of IMP2 during the progression of liver tumors, transgenic zebrafish lines Tg(fabp10:tetoff; tre:imp2/gfp) with constitutively zebrafish IMP2 and GFP-overexpressed in the liver had been established in our laboratory by using liver-specific LFABP promoter combined with TetOff bi-directional expression system via Tol2 transposon-mediated transgenesis. The liver-specific IMP2 transgenic zebrafish stably inherited to F5 generation will develop severe intrahepatic cholangiocarcinoma (ICC) at around 10-week old. Pathological progression of ICC in IMP2 transgenic zebrafish was revealed from liver injury without cholangiocyte proliferation at 4-week old to cholangiocyte proliferation at 6-week old then progressing to ICC formation at around 10-week old zebrafish. Transgenic IMP2 expression was turned off by tetracycline treatment (1μg/ml) from 1-week to 6-week old. There was no intrahepatic cholangiocyte proliferation observed in IMP2 transgenic zebrafish treated with tetracycline at 6-week old. The result confirmed that intrahepatic cholangiocyte proliferation in IMP2 transgenic zebrafish is an IMP2-dependent phenotype. To study the early stage mechanism of IMP2-induced ICC, the liver with abnormal hepatocytes of 4-week IMP2 transgenic fish was compared with normal liver of control transgenic fish Tg(fabp10:tetoff; tre:gfp) by microarray analysis and evaluated by Q-PCR. We found the critical antocrine/paracrine signaling genes including IGF-IIa, IGF-2b, IGF-IRa, IGF-IRb and Progranulin A (PGRN-A) were all up-regulated in the liver of 4-week IMP2 transgenic zebrafish. The results indicate liver-specific IMP2 overexpression may activate IGF-II and progranulin signalling to stimulate cholangiocyte proliferation and increase PTEN and AKT phosphorylation to attenuate PTEN and activate AKT-mediated anti-apoptosis signal in the early stage of ICC progression. Furthermore, the antisense vivo-Morpholinos of IGF-2b, IGF-IRa, IGF-IRb and PGRN-A were used to treat IMP2 transgenic zebrafish from 3-week to 6-week old by intraperitoneal (I.P.) injection, respectively. Inhibition of IGF-IRa and IGF-IRb by vivo-Morpholino (Mo) can significantly reduce intrahepatic cholangiocyte proliferation from 78% (control: PBS injection) to 50% and 44%, respectively. Inhibition of IGF-II and PGRN-A by vivo-Mophonino can also reduce intrahepatic cholangiocyte proliferation from 78% to 64% and 75%, respectively. The data indicates that IGF-IRs play a critical role in cholangiocyte proliferation during the early stage of ICC progression induced by IMP2 overexpression. Effective blockage of IGF-IR signaling with vivo-Mophonino may provide the evidence of using IGF-IR signaling as therapeutic target for early diagnosis and/or treatment of ICC. This liver-specific IMP2-induced ICC transgenic zebrafish provides a novel animal model to study the interaction of hepatocytes and cholangiocytes in ICC progression.
|Appears in Collections:||[水產養殖學系] 博碩士論文|
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