2015-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/644775摘要：台灣地區除了B型肝炎帶原者眾多，肝硬化、慢性肝病、及肝癌發生率很高，近年來脂肪肝患者也急劇升高，肝臟疾病已是台灣地區十大死因前2名，對國人健康的威脅相當大。有關正常肝臟在肝損傷後可以再生，已有許多研究證實，多種生長因子、酵素、賀爾蒙、藥劑、營養素等因素也被證明，可直接或間接影響肝臟再生之啟動、分化、終結等基因調控機轉。脂肪肝雖然被認為是良性病症，但脂肪肝後續的脂肪變性與纖維化，在肝損傷與肝切除情況下，會影響肝再生並導致病患死亡或肝臟功能失常機率增加。探討脂肪肝肝臟再生關鍵基因之表現及其調控機轉，已是肝臟再生領域的重要議題。我們以前研究，以基因微陣列監控，發現肝臟再生過程中基因表現，依時間序共有72種變化型態，其中細胞凋亡、血管新生、免疫反應等相關基因之表現，以Bayesian及Boolean Network電腦程式的分析，均呈現高度有意義之變化；且在時間序及程度上皆反應出與肝臟再生有高度密切的關係，同時這些高度變遷之基因表現，互相間亦有相當密切的關連性。我們近期的研究成果顯示: 肝臟部份切除後，抗細胞凋亡蛋白質(Mcl-1L)會受免疫調控激素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)。IL-6也會調控肝臟細胞產生血管新生相關因子angiotensinogen之表現，可能在血壓的調控上扮演角色，並對於肝臟再生中，血液再灌流的效應有重要的臨床意義(發表於PLOS One, 2013)。我們也發現具有innate immunity調控作用的Lipocalin-2 (LCN2)蛋白質，在肝臟再生過程中會被大量表現，顯示免疫機制在肝臟再生過程中的重要性(已發表於Int J Surg. 2013)。有關脂肪肝的研究，我們初步証實，脂肪肝之細胞凋亡(TUNEL及caspase 3)在肝臟部份切除後會明顯增加，並造成肝臟再生明顯較差。然而，脂肪肝之肝臟再生分子機轉目前仍不清楚。本計畫將探討，脂肪肝部分肝臟切除後，肝臟再生過程中，細胞凋亡、血管新生、免疫反應等相關基因之表現。我們將使用methionine and choline deficient (MCD) diet誘發脂肪肝之小鼠，接受百分之七十及百分之四十肝臟部分切除手術，於術前及術後2、4、6、12、24、48、72小時及5、7天犧牲取樣，探討:(1)細胞凋亡(第一年)；(2)血管新生(第二年)；及(3)免疫反應(第三年)相關基因表現之變化，釐清及確認脂肪肝肝臟再生過程中的關鍵基因表現模式，及其對肝臟再生程度之影響，並研擬脂肪肝肝臟再生之相關訊息傳遞路徑及其機轉。分三年的研究策略與方法如下:第一年：鑑定及確認脂肪肝肝臟再生過程中，細胞凋亡之關鍵基因及相關基因的變化，分析其訊息傳遞路徑，並描繪出肝臟再生時，細胞凋亡相關基因之調控路徑及機轉。將進行：(1) 脂肪肝部份切除後，剩餘肝臟之重量比值，並計算剩餘肝臟之有絲分裂指標mitotic index。(2) 以BioCata-Charting Pathways of Life, BioCata website，或論文檢索方式，搜尋細胞凋亡相關基因的特性及其與肝臟再生之關聯，並選出具代表性之細胞凋亡關鍵基因(如casp 3、casp 12、Lcn2、Bcl-2、Mc1-1及其他相關基因等)，驗證其是否為脂肪肝肝臟再生高度相關之強力相關基因(strongly related gene)。(3) 以基因微陣列高密度基因晶片(20,500點)、肝細胞mRNA標號、hybridization及影像分析等方法，測定剩餘肝臟內，各種細胞凋亡之關鍵基因[key gene：基因變遷程度須為(a) 70%切肝大於40%切肝；(b) 70%切肝平行40%切肝；(c)變化程度大於2倍(上升)表 C011 共 3 頁 第 2 頁或低於二分之一(下降)者，以Array-Pro image acqnisition software及Splus 6.1分析軟體找尋，細胞凋亡相關基因等]表現之變遷。(4) 以Q-PCR確認各細胞凋亡關鍵基因mRNA表現之變遷，是否與基因表現平行。(5) 以Western Blot及免疫生化染色法確認各細胞凋亡關鍵基因蛋白產量之變遷，以確認其為肝臟再生之關鍵基因。(6) 以siRNA抑制法測定肝細胞再生過程中，細胞凋亡關鍵基因之表現，是否被抑制，以確認其訊息傳遞路徑在脂肪肝肝臟再生過程中扮演之角色。(7) 以特殊Bayesian and Boolean Network電腦程式（含環狀、線性、隨機取樣等三種方法）、及Splus 6.1分析軟體，分析所有選定並確認之細胞凋亡關鍵基因，及其強力相關基因，在肝臟再生過程中基因表現程度、型態、與時程相關之變遷，並分析其在脂肪肝各細胞凋亡關鍵基因調控路徑之角色。(8) 依各細胞凋亡關鍵基因特性及其與肝臟再生之關聯，嘗試描繪出脂肪肝肝臟再生時，細胞凋亡關鍵基因及強力相關基因之完整基因訊息傳遞路徑，並比較正常肝及脂肪肝肝臟再生過程中，有關啟動、分化及終結之基因調控路徑及機轉的差異。第二年：鑑定及確認脂肪肝肝臟再生過程中，血管新生之關鍵基因及相關基因的變化，分析其訊息傳遞路徑，並描繪出肝臟再生時，血管新生相關基因之調控路徑及機轉。將進行：(1) 脂肪肝部份切除後，剩餘肝臟之重量比值，並計算剩餘肝臟之有絲分裂指標mitotic index。(2) 以BioCata-Charting Pathways of Life, BioCata website，或論文檢索方式，搜尋血管新生相關基因的特性及其與肝臟再生之關聯，並選出具代表性之血管新生關鍵基因(如Agtn、At1、Fkbp12、p38及其他相關基因等)，驗證其是否為脂肪肝肝臟再生高度相關之強力相關基因(strongly related gene)。(3) 以基因微陣列高密度基因晶片(20,500點)、肝細胞mRNA標號、hybridization及影像分析等方法，測定剩餘肝臟內，各種血管新生之關鍵基因[key gene：基因變遷程度須為(a) 70%切肝大於40%切肝；(b) 70%切肝平行40%切肝；(c)變化程度大於2倍(上升)或低於二分之一(下降)者，以Array-Pro image acqnisition software及Splus 6.1分析軟體找尋，血管新生相關基因等]表現之變遷。(4) 以Q-PCR確認各血管新生關鍵基因mRNA表現之變遷，是否與基因表現平行。(5) 以Western Blot及免疫生化染色法確認各血管新生關鍵基因蛋白產量之變遷，以確認其為肝臟再生之關鍵基因。(6) 以siRNA抑制法測定肝細胞再生過程中，血管新生關鍵基因之表現，是否被抑制，以確定其訊息傳遞路徑，在脂肪肝肝臟再生過程中扮演之角色。(7) 以特殊Bayesian and Boolean Network電腦程式（含環狀、線性、隨機取樣等三種方法）、及Splus 6.1分析軟體，分析所有選定並確認之血管新生關鍵基因，及其強力相關基因，在血管新生過程中基因表現程度、型態、與時程相關之變遷，並分析其在脂肪肝各血管新生關鍵基因調控路徑之角色。(8) 依各血管新生關鍵基因特性及其與肝臟再生之關聯，嘗試描繪出脂肪肝肝臟再生時，血管新生關鍵基因及強力相關基因之完整基因訊息傳遞路徑，並比較正常肝及脂肪肝表 C011 共 3 頁 第 3 頁肝臟再生過程中，有關啟動、分化及終結之基因調控路徑及機轉。第三年：鑑定及確認脂肪肝肝臟再生過程中，免疫反應相關之關鍵基因及相關基因的變化，分析其訊息傳遞路徑，並描繪出肝臟再生時，免疫反應相關基因之調控路徑及機轉。將進行：(1) 脂肪肝部份切除後，剩餘肝臟之重量比值，並計算剩餘肝臟之有絲分裂指標mitotic index。(2) 以BioCata-Charting Pathways of Life, BioCata website，或論文檢索方式，搜尋免疫反應相關基因的特性及其與肝臟再生之關聯，並選出具代表性之免疫反應相關基因(如IFNγR、IL-6、Orm-1、Nfe-2(Nrf2)及其他相關基因等)，驗證其是否為脂肪肝肝臟再生高度相關之強力相關基因(strongly related gene)。(3) 以基因微陣列高密度基因晶片(20,500點)、肝細胞mRNA標號、hybridization及影像分析等方法，測定剩餘肝臟內，各種免疫反應之關鍵基因[key gene：基因變遷程度須為(a) 70%切肝大於40%切肝；(b) 70%切肝平行40%切肝；(c)變化程度大於2倍(上升)或低於二分之一(下降)者，以Array-Pro image acqnisition software及Splus 6.1分析軟體找尋，免疫反應相關基因等]表現之變遷。(4) 以Q-PCR確認各免疫反應相關關鍵基因mRNA表現之變遷，是否與基因表現平行。(5) 以Western Blot及免疫生化染色法確認各免疫反應相關關鍵基因蛋白產量之變遷，以確認其為肝臟再生之關鍵基因。(6) 以siRNA抑制法測定肝細胞再生過程中，免疫反應相關關鍵基因之表現，是否被抑制，以確認其訊息傳遞路徑，在脂肪肝肝臟再生過程中扮演之角色。(7) 以特殊Bayesian and Boolean Network電腦程式（含環狀、線性、隨機取樣等三種方法）、及Splus 6.1分析軟體，分析所有選定並確認之免疫反應相關關鍵基因，及其強力相關基因，在肝臟再生過程中基因表現程度、型態、與時程相關之變遷，並分析其在脂肪肝各免疫反應相關關鍵基因調控路徑之角色。(8) 依各免疫反應相關關鍵基因特性及其與肝臟再生之關聯，嘗試描繪出脂肪肝肝臟再生時，免疫反應相關關鍵基因及強力相關基因之完整基因訊息傳遞路徑，並比較正常肝及脂肪肝肝臟再生過程中，有關啟動、分化及終結之基因調控路徑及機轉。綜合三年研究成果，期能發現脂肪肝肝臟再生過程中，細胞凋亡、血管新生及免疫反應關鍵基因及強力相關基因之完整基因訊息傳遞路徑，並比較正常肝及脂肪肝肝臟再生過程中，有關啟動、分化及終結之基因調控路徑及機轉。以便進一步做為脂肪肝肝臟部份切除後，肝臟再生基因治療的根據。<br> Abstract: Because of the high incidence of hepatitis B carrier and hepatoma patients, and also patients with fatty liver (FL), the mortality rate of hepatic disease is one of the tope ten causes of death in Taiwan. Although there are much controversy on the initiation, differentiation and termination of liver regeneration (LR), several factors such as enzyme, growth factor, hormone, pharmacological agent, and nutrition have been demonstrated to directly or indirectly affect LR. Although FL is often considered to be a benign condition, steatosis is proved to have higher frequency of mortality and morbidity after liver resection. The study of gene-control mechanism during LR in FL patients has become an important issue.In our previous study by mass survey with cDNA microarray, we have found 72 different patterns of gene expression during LR after partial hepatectomy (PH). By Bayesian and Boolean Network computer program analysis, we have demonstrated that the expressions of apoptosis, angiogenesis, and immune related genes have strong correlations to LR after PH. Our recent studies revealed that anti-apoptosis protein Mcl-1L is regulated by immune modulating cytokine IL-6 and is critically involved in the regeneration of hepatocytes (PLOS One, 2013). We also found that IL-6 could regulate the angiotensinogen (angiogenesis-related protein) expression during liver regeneration; it may play roles on the blood pressure regulation and on the reperfusion during LR (PLOS One, 2013). We also demonstrated that Lipocalin-2 (LCN2) protein which has role in innate immunity is expressed markedly during LR (Int J Surg. 2013). In our previous study, we have demonstrated apoptosis cell (by TUNEL and caspase 3) increased markedly during LR after PH in FL, which decreased LR also noted significantly. However, the expressions of apoptosis, angiogenesis, and immune related genes are not well defined during LR after PH in FL.In this project, we will detect the variations in expressions of apoptosis, angiogenesis, and immune related key genes during LR after PH in FL mice. Mice with MCD induced FL will be used as subject. PH around 70% and 40% will be performed and they will be sacrificed before and at 2, 4, 6, 12, 24, 48, 72 hours and 5, 7days after hepatectomy. We defined the key genes in the gene-control mechanism of LR as follows: the variations of key genes expressions should be: (1) stronger in 70% PH than 40% PH; (2) parallel between 70% PH and 40% PH; and (3) the elevation is higher than twice (up-regulated) or less than half (down-regulated) of prehepatectomized level. We will measure the variations in expressions of apoptosis, angiogenesis, and immune related key genes during LR after PH in FL:表 C011 共 4 頁 第 2 頁The 1st Year Protocol: Survey and verify the apoptosis related key genes, analyze all concerned gene-signaling pathways, and figure out apoptosis related gene control pathways and the mechanism during LR in FL. The following methods will be performed:(1) Measure the weight of remnant liver, and the mitotic index in the remnant liver after PH in FL.(2) Search for the gene-control pathways which are related to the apoptosis related key genes by BioCata-Charting Pathways of Life, BioCata Web, journal searchings; selecting the apoptosis strongly related genes (such as casp 3, casp 12, Lcn2, Bcl-2, Mcl-1, and other related genes etc.) from the concerned gene-control pathways; detect the variations of these genes expressions and verifying if they are the strongly related genes during LR after PH in FL.(3) Survey the genomic the apoptosis related genes expressions, by cDNA microarray, labeling of mRNA hybridization, and image analysis of the apoptosis related key genes as described in above section.(4) Verify the mRNA expression of the apoptosis related key genes by real time polymerase chain reaction (Q-PCR), and analyze the compatibility between the genes expressions and the mRNA expression.(5) Verify the protein products of the apoptosis related key genes by Western Blot and immunohistochemical stain, and analyze the compatibility between the genes expressions and the protein products to define if they are the key genes.(6) Inhibition of these apoptosis related genes expressions by siRNA, to evaluate if they are inhibited, and identify their signaling pathway during LR in FL.(7) Analyze all the key genes detected and verified as the apoptosis strongly related genes by specific Bayesian and Boolean Network computer program (including circular, layer layout and randomized pattern), and Splus 6.1 analysis software, to find out the changing patterns, degrees, and time sequences of all genes and the relationships with LR in FL.(8) Figure out an apoptosis concerned gene-signaling pathway for LR in FL. After fully understanding the roles of apoptosis related key genes according to the genes’ characteristics and their relationship to LR, we will compare the changes of apoptosis genes pattern between the FL and normal liver as our previous study.The 2nd Year Protocol: Survey and verify the angiogenesis related key genes, analyze all concerned gene-signaling pathways, and figure out angiogenesis related gene control pathways and the mechanism during LR in FL. The following methods will be performed:表 C011 共 4 頁 第 3 頁(1) Measure the weight of remnant liver, and the mitotic index in the remnant liver after PH in FL.(2) Search for the gene-control pathways which are related to the angiogenesis related key genes by BioCata-Charting Pathways of Life, BioCata Web, journal searchings; selecting the angiogenesis strongly related genes (such as Agtn, At1, Fkbp12, p38, and other related genes etc.) from the concerned gene-control pathways; detect the variations of these genes expressions and verifying if they are the strongly related genes during LR after PH in FL.(3) Survey the genomic of the angiogenesis related genes expressions, by cDNA microarray, labeling of mRNA hybridization, and image analysis of the angiogenesis related key genes as described in above section.(4) Verify the mRNA expression of the angiogenesis related key genes by real time polymerase chain reaction (Q-PCR), and analyze the compatibility between the genes expressions and the mRNA expression.(5) Verify the protein products of the angiogenesis related key genes by Western Blot and immunohistochemical stain, and analyze the compatibility between the genes expressions and the protein products to define if they are the key genes.(6) Inhibition of these angiogenesis related genes expressions by siRNA, to evaluate if they are inhibited, and identify their signaling pathways during LR in FL.(7) Analyze all the key genes detected and verified as the angiogenesis strongly related genes by specific Bayesian and Boolean Network computer program (including circular, layer layout and randomized pattern), and Splus 6.1 analysis software, to find out the changing patterns, degrees, and time sequences of all genes and the relationships with LR in FL.(8) Figure out an angiogenesis concerned gene-signaling pathway for LR in FL. After fully understanding the roles of angiogenesis key genes according to the genes’ characteristics and their relationship to LR, we will compare the changes of angiogenesis related genes pattern between the FL and normal liver as previous study.The 3rd Year Protocol: Survey and verify the immune related key genes, analyze all concerned gene-signaling pathways, and figure out immune related gene control pathways and the mechanism during LR in FL. The following methods will be performed:(1) Measure the weight of remnant liver, and the mitotic index in the remnant liver after PH in FL.(2) Search for the gene-control pathways which are related to the immune表 C011 共 4 頁 第 4 頁related key genes by BioCata-Charting Pathways of Life, BioCata Web, journal searchings; selecting the immune strongly related genes (such as IFNγR, IL-6, Orm-1, Nfe-2(Nrf2), and other related genes etc.) from the concerned gene-control pathways; detect the variations of these genes expressions and verifying if they are the strongly related genes during LR after PH in FL.(3) Survey the genomic of the immune related genes expressions, by cDNA microarray, labeling of mRNA hybridization, and image analysis of the immune related key genes as described in above section.(4) Verify the mRNA expression of the immune related key genes by real time polymerase chain reaction (Q-PCR), and analyze the compatibility between the genes expressions and the mRNA expression.(5) Verify the protein products of the immune related key genes by Western Blot and immunohistochemical stain, and analyze the compatibility between the genes expressions and the protein products to define if they are the key genes.(6) Inhibition of these immune related genes expressions by siRNA, to evaluate if they are inhibited, and identify their signaling pathway during LR in FL.(7) Analyzing all the key genes detected and verified as the immune strongly related genes by specific Bayesian and Boolean Network computer program (including circular, layer layout and randomized pattern), and Splus 6.1 analysis software, to find out the changing patterns, degrees, and time sequences of all genes and the relationships with LR in FL.(8) Figure out an immune concerned gene-signaling pathway for LR in FL. After fully understanding the roles of immune related key genes according to the genes’ characteristics and their relationship to LR, we will compare the changes of immune related genes pattern between the FL and normal liver as our previous study.Taken together this 3-year project may help to confirm the apoptosis, angiogenesis and immune related gene-control mechanisms of initiation, differentiation and termination during LR in FL. We may also detect the difference of the gene-control pathway between FL and normal liver, and that may suggest a good way to offer genetherapy for a better LR after PH in FL patients.脂肪肝肝臟再生肝臟部份切除細胞凋亡血管新生免疫反應關鍵基因強力相關基因基因微陣列Western BlotQ-PCR免疫生化染色法siRNA抑制基因調控路徑fatty liver (FL)liver regeneration (LR)partial hepatectomy (PH)apoptosisangiogenesisimmunekey genestrongly related genemicroarrayWestern BlotRT-PCRimmunohistochemical stainsiRNA inhibitiongene-control pathway