摘要:肝臟部份切除後,肝臟細胞再生及凋亡調控機制的平衡,在肝臟再生過程中,扮演著重要角色。我們最新發表的研究成果顯示,肝臟部份切除後,肝臟再生過程中肝藏組織內與調控細胞凋亡有關的 Lcn2 蛋白會被表現(已發表於 Int J Surg. 2013);抗細胞凋亡蛋白質(Mcl-1L)則會受 IL-6調控而表現,對肝臟細胞凋亡扮演重要功能(發表於 PLOS One, 2013);我們也發現 IL-6會調控肝臟細胞產生 Angiotensinogen,可能在血壓的調控上扮演角色 (發表於PLOS One, 2013)。然而肝臟再生過程中,除了肝細胞,肝臟內之非肝細胞(如Kupffer cells, sinusoidal endothelial cells, stellate cells等)也會再生,其相關調控機轉研究則尚少。此外,當病患併有脂肪肝,很可能影響肝臟再生程度,甚而降低病患存活率,此方面之基因機轉研究也仍甚少。 本計畫我們將延續過去,利用小鼠肝臟部份切除之動物模式,探討抗凋亡蛋白Mcl-1L在(1)正常肝及(2)脂肪肝之肝臟再生過程中,肝細胞及非肝細胞之調控機制,並研究其基因治療之應用。 第一年:區別Mcl-1L mRNA及其蛋白質在肝細胞及非肝細胞之表現;並利用細胞學研究探討Mcl-1L相關基因之表現與調控機制: A. 以切肝後剩餘肝臟之有絲分裂指標(mitotic index)、Ki67 staining、TUNEL assay確認肝臟再生及凋亡程度。 B. 以免疫組織染色法研究 Mcl-1L 在肝細胞及非肝細胞表現及相關基因之調控機轉。 C. 以 Percoll gradient技術,分離並純化肝臟內肝細胞及非肝細胞,進一步運用化學抑制劑及 decoy ODN 技術,探討誘發肝細胞及非肝細胞產生Mcl-1L訊息之傳遞路徑,並比較其是否有一致性。 D. 運用 siRNA 基因表現抑制技術,抑制肝細胞及非肝細胞 Mcl-1L基因表現,再以流式細胞儀配合 Ki67/Annexin-V/PI 染色,鑑定其對肝細胞及非肝細胞在生長、凋亡及細胞週期之效應,並比較其是否有一致性。 第二年:利用我們已經建立的 methionine and choline deficient (MCD) diet誘發小鼠脂肪肝的模式,探討脂肪肝肝臟再生過程中,肝細胞及非肝細胞Mcl-1L mRNA及其蛋白質在脂肪肝情況下,各類細胞再生的表現,並研究其相關基因調控機制。 A. 確認脂肪肝之肝臟再生、凋亡程度(方法同第一年之 A),並比較其與正常肝臟再生程度之差異性。 B. 確認Mcl-1L在脂肪肝之肝臟再生過程中,相關基因表現及其調控機轉。 (方法同第一年之 B),並比較其與正常肝臟再生過程之差異性。 C. 探討脂肪肝誘發肝細胞及非肝細胞產生Mcl-1L訊息之傳遞路徑(方法同第一年之 C),並比較其與正常肝臟再生過程之差異性。 D. 鑑定脂肪肝肝臟再生過程中,肝細胞及非肝細胞在生長、凋亡及細胞週期之效應(方法同第一年之 D) ,並比較其與正常肝臟再生過程之差異性。 第三年:利用我們已經建立之誘導式肝臟組織專一性 Mcl-1L 基因剔除(knockdown)小鼠與脂肪肝小鼠模式,探討Mcl-1L基因治療對肝臟再生之療效,並探討肝臟再生過程中 Mcl-1L 與免疫、凋亡、血管新生相關基因的調節機制: A. 以肝細胞專一性 Mcl-1L基因剔除小鼠(具有肝臟細胞 albumin promoter調控Mcl-1L shRNA之特性),研究Mcl-1L調控肝臟再生的直接証據。 B. 利用 Mcl-1L 基因治療技術,用於 Mcl-1L 基因剔除小鼠,研究肝臟再生過程中,肝細胞及非肝細胞之療效。(以第一、二年之方法 A、B、C、D定量分析成果),並比較其與正常肝臟再生過程之差異性。 C. 利用基因治療技術,研究脂肪肝小鼠,肝臟再生過程中,肝細胞及非肝細胞之療效(方法同第三年之 A、B),並比較其與正常肝臟再生過程之差異性。
Abstract: The balances between cell proliferation and apoptosis play important role in liver regeneration (LR) after liver partial hepatectomy (PH). Our recent studies revealed that Lcn2 protein is expressed during LR (published Int J Surg. 2013), anti-apoptosis protein Mcl-1L is regulated by IL-6 and is critically involved in the regeneration of hepatocytes(published in PLOS One, 2013). We also found that IL-6 could regulate the angiotensinogen expression during LR; it may play roles on the blood pressure regulation (published in PLOS One, 2013). However, during LR, non-hepatocytes; such as Kupffer cells, sinusoidal endothelial cells, stellate cells, as well as hepatocytes are also proliferative, but the regulatory mechanism is not clear. Additionally, fatty liver may attenuate the LR and decreases survival rate of liver injury including PH. In this project, we will follow the past experimental experiences, using the liver PH technique to survey and verify the regulation and the role of anti-apoptotic gene Mcl-1 during LR in both hepatocytes and non-hepatocytes, and in fatty liver mice, to evaluate the therapeutic effect of Mcl-1L gene therapy on enhancing LR in normal liver or fatty liver. The study designs and strategies of each year are: The first year: To compare the Mcl-1L mRNA and protein expression in hepatocytes and non-hepatocytes during LR after PH; and to evaluate the Mcl-1L gene expression and regulation by molecular cell biology: A. To identify the cell proliferation status of the remnant liver by mitotic index and Ki67 staining; and to clarify the regeneration and apoptosis phenomena by TUNEL assay. B. To determine the Mcl-1L and its related protein expression in hepatocytes and non-hepatocytes by IHC. C. To isolate mouse hepatocytes and non-hepatocytes by Percoll gradient assay; and to identify and compare the critical signaling pathways and transcriptional factors that involved in the Mcl-1L regulation by chemical inhibitors and decoy ODN strategy. D. To inhibit the Mcl-1L gene expression by siRNA strategy in hepatocytes and non-hepatocytes; to evaluate the effect of Mcl-1L expression on cell growth, apoptosis, and cell cycle by Ki67/Annexin-V/PI staining based flow cytometric analysis. The second year: To investigate the fatty liver mouse model by methionine and choline deficient (MCD) diet feeding protocol. To compare the Mcl-1L mRNA and protein expression in hepatocytes and non-hepatocytes during LR after PH; and to evaluate the Mcl-1L gene expression and regulation by molecular cell biology: A. To identify the LR of the fatty liver model (with the same methodologies described in the part A of the first year); to compare the differences of LR between normal and fatty liver models. B. To determine the Mcl-1L and its related protein expression in hepatocytes and non-hepatocytes in fatty liver model (with the same methodologies described in the part B of the first year); to compare the differences of LR between normal and fatty liver models. C. To compare and identify the critical signaling pathways and transcriptional factors that involved in the Mcl-1L regulation in both types of cells in fatty liver model; to compare the differences of LR between normal and fatty liver models. D. To evaluate the effect of Mcl-1L expression on cell growth, apoptosis, and cell cycle in hepatocytes and non-hepatocytes in fatty liver model; to compare the differences of LR between normal and fatty liver models. The third year: To investigate the therapeutic effect of Mcl-1L gene therapy on liver-specific Mcl-1L conditional knockdown mice and fatty liver mice. Based on these two animal models, we will also evaluate the regulation of Mcl-lL related immunity, apoptosis and angiogenesis during the LR after PH: A. To establish the inducible and liver specific Mcl-1L conditional knockdown mice, which based the tetracycline on for inducible and albumin promoter for liver specific. The gene knockdown mice would apply the direct evidence to confirm the role of Mcl-1L during LR. B. To evaluate the therapeutic effect of Mcl-1L gene therapy on liver specific Mcl-1L conditional knockdown mice with PH and to evaluate the effect in hepatocytes and non-hepatocytes, and to compare the differences of LR between normal and gene therapy models. C. To evaluate the therapeutic effect of Mcl-1L gene therapy on fatty liver mice with PH, both in hepatocytes and non-hepatocytes, and to compare the differences of LR between normal and gene therapy models.