摘要:肝臟再生過程中,基因調控機轉的研究,近年來已成為醫界最重要的研究議題之一。由於台灣地區B型肝炎帶原者眾多,肝硬化、慢性肝病、及肝癌發生率很高,肝癌加上肝病死亡人數,已是台灣地區十大死因之首,肝病對國人健康的威脅相當大。有關肝細胞在肝損傷後可以再生,雖已是公認的事實,許多研究也証實多種生長因子、酵素、賀爾蒙、藥劑、營養素等,可直接或間接影響肝臟再生,但肝臟再生之啟動、分化、終結等基因調控機轉,則仍不明瞭。肝臟部分切除後,肝臟細胞再生及細胞凋亡現象均十分明顯,許多細胞凋亡相關基因表現在再生過程中,可能扮演十分重要之角色,而細胞凋亡與血管新生及免疫反應之基因訊息傳遞均有關連性,但其間基因調控路徑相關研究之報告並不多見。Fukuhara等人發現,肝臟部分切除後早期,許多基因族群之表現,強烈與再生現象相輔相成。Schleimer等人研究証實,細胞凋亡之增加可抑制肝臟再生。Vick等人也發現抗凋亡(anti-apoptotic)基因Mc-1被去除(knock out)後,肝細胞凋亡會增加,肝臟再生則明顯減少,顯示細胞凋亡基因與肝臟再生息息相關。Sookoian等人研究証實血管新生相關基因angiotensinogen (Agtn)與肝臟再生相關,Liu等人則証實Agtn基因表現與細胞凋亡息息相關。免疫反應相關基因如IFM-γ、CD4+Th1等,也被証實與肝臟再生相關,同時與細胞凋亡訊息傳遞也相關。Isomoto等人之研究証實,IL-6基因可經由STAT3基因調控路徑,正向調控Mc-1抗凋亡基因,進而影響肝細胞凋亡與再生。以基因微陣列(microarray)全面監控肝臟再生關鍵基因之表現及研究基因調控路徑之機轉,則尚未有結果報告。吾人以基因微陣列監控肝臟再生,發現肝臟再生過程中,基因表現之變化,依時間序共有72種變化型態,其間細胞凋亡、血管新生、免疫反應等相關基因之表現,均呈現相當有意義之變化,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。吾人再以Bayesian及Boolean Network電腦程式分析,發現這些高度變遷之基因表現,互相間亦有相當密切的關連性。肝臟再生的速度在70%部分切肝時,明顯比40%部分切肝快速。基於調控肝臟再生關鍵基因之變遷,也應相對有較明顯之變遷,吾人於本研究初期,以cDNA基因微陣列及Array-Pro image acquisition software以及Splus 6.1統計軟體,分析基因微陣列全面監控下,搜尋出基因變遷型態及程度,需合乎:(1)70%切肝大於40%切肝;(2)70%切肝平行40%切肝;(3)變化程度大於2倍(上升)或低於二分之一(下降)者,定義為肝臟再生相關基因,初步發現細胞凋亡、血管新生、及免疫反應相關基因之表現,與肝臟再生關係特別密切,分別如: (A)細胞凋亡相關基因:caspase 3 (casp 3)、caspase 12 (casp 12)、Lipocalin-2 (Lcn2)、B-cell lymphoma 2 (Bcl-2)、myeloid cell leukemia-1(Mc1-1);(B)血管新生相關基因:angiotensinogen (Agtn)、angiotensin Ⅱ type 1A recaptor(At1)、fatty acid binding protein 12 (Fkbp12)、p38; 及(C)免疫反應相關基因: Interferon gamma recaptor (IFNγR)、interleukin 6 (IL-6)、Orosomucoid 1 (Orm-1)、NF-E2-related factor 2 (Nfe-2, Nrf2)等。這些基因是否為肝臟再生之關鍵基因?其基因調控訊息路徑為何?相互間是否有相互影響之基因調控路徑?均仍不明瞭,應進一步作:①基因表現之確認(Q-PCR, Western blot, 免疫生化染色等);②以siRNA抑制基因表現法鑑定該基因與肝臟再生之相關性;③由BioCarta相關網站或專業論文上,搜尋細胞凋亡、血管新生、及免疫反應相關之基因調控路徑;④再由路徑上找出各個強力相關基因,鑑定及再確認其基因表現之變遷,證明其與肝臟再生是否息息相關;⑤再藉由所有搜尋並鑑定確認之細胞凋亡、血管新生、及免疫反應關鍵基因與強力相關基因之變遷,嘗試描繪出肝臟再生之基因調控路徑及機轉。本計劃擬分三年進行,將以重約200克之Wistar雄性大鼠作實驗,分別接受百分之七十及百分之四十肝臟部分切除手術,各於術前及術後2、4、6、12、24、48、72小時及5、7天犧牲取樣,測定:(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,鑑定及確認該基因與肝臟新生之相關性,並搜尋及鑑定其相關訊息傳遞路徑:第一年:鑑定及確認肝臟再生過程中,細胞凋亡之關鍵基因及相關基因(1) 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。,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:(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)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,嘗試描繪出肝臟再生時細胞凋亡關鍵基因→強力相關基因之完整基因訊息傳遞路徑,期能更深入証實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。第二年:鑑定及確認肝臟再生過程中,血管新生之關鍵基因及相關基因(1) 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。,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:(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,嘗試描繪出肝臟再生時細胞凋亡及血管新生關鍵基因→強力相關基因之完整基因訊息傳遞路徑,期能更深入証實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:鑑定及確認肝臟再生過程中,免疫反應相關之關鍵基因及相關基因(1) 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。,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:(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,嘗試描繪出肝臟再生時細胞凋亡、血管新生及免疫反應相關關鍵基因→強力相關基因之完整基因訊息傳遞路徑,期能更深入証實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。
Abstract: Because of the high incidence of hepatitis B carrier and hepatoma patients, 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, several factors such as enzyme, growth factor, hormone, pharmacological agent, and nutrition have been demonstrated to directly or indirectly affect liver regeneration. The study of gene-control mechanism during liver regeneration has become an important issue in recent years. Fukuhara et al reported that cluster genes had strong correlations with liver regeneration at early stage after partial hepatectomy (PH). Schleimer K et al have demonstrated that portal vein arterializations increases hepatocellular apoptosis and inhibits liver regeneration. Vick B et al have shown that knock out of anti-apoptotic gene Mcl-1 can induce liver damage and increase apoptosis susceptibility of murine hepatocytes. Sookoian S et al have proved that angiogenesis correlated gene, angiotensinogen (Agtn) is related to both angiogenesis and apoptosis. Agtn may inhibit angiogenesis by inducing apoptosis of endothelial cells. Agtn may also enhance apoptosis in various cell types, including alveolar cells, cardiomyocytes, and renal tubular cells. These lines of evidence suggest that Agtn may play an essential role in the process of liver regeneration. Immune response related gene, such as IFN-γ which is a dimeric glycoprotein that has many immune regulatory functions and can induce cell proliferation and apoptosis. It is secreted from CD4+Th1 and CD8 cells, and mainly synthesized by NK cells and activated T cells, was proved to be correlated with liver regeneration. IL-6 releases from Kupffer cells followed by STAT3 activation in hepatocytes contributes to liver regeneration after PH. Moreover, IL-6 was proved to up-regulate Mcl-1 expression through a STAT3 pathway. Our previous studies demonstrated that immune, angiogenesis, growth factor, enzyme, and hormone related genes expressions have marked changes during liver regeneration. By mass survey with cDNA microarray, we have found 72 different patterns of gene expression during liver regeneration. By Bayesian and Boolean Network computer program analysis, we also demonstrated that the expressions of apoptosis, angiogenesis, and immune related genes have unique strong correlations to liver regeneration.The regenerative degree is higher in 70% PH than in 40% PH. The variations of key genes expressions in the gene-control pathway should also be stronger in 70% PH than 40% PH. By cDNA microarray and Array-Pro imageacquisition software and Splus 6.1 statistics software, we preliminarily detected some apoptosis, angiogenesis, and immune related key genes which are highly compatible to liver regeneration: (A) Apoptosis related key genes: caspase 3 (casp 3), caspase 12 (casp 12), Lipocalin-2 (Lcn2), B-cell lymphoma 2 (Bcl-2), myeloid cell leukemia-1 (Mcl-1); (B)Angiogenesis related key genes: angiotensinogen (Agtn), angiotensin II type 1A receptor (At1), fatty acid binding protein 12 (Fkbp12), p38; and (C)Immune related key genes: Interferon gamma receptor (IFNγR), interleukin 6 (IL-6), Orosomucoid 1 (Orm-1), NF-E2-related factor 2 (Nfe-2, Nrf2). However, did they truly play as the key genes during liver regeneration? Are there other strongly related genes of apoptosis, angiogenesis, and immune involved? And, what are their gene-signaling pathways? Further verification of key genes expressions by: ①Q-PCR, Western Blot and immunohistochemical stain for mRNA expressions and protein products; ②siRNA inhibition of these related genes expressions to identify their roles in liver regeneration; ③Searching for apoptosis, angiogenesis, and immune related gene-signaling pathways; ④Selecting the strongly related genes of apoptosis, angiogenesis, and immune key genes from the concerned gene-signaling pathways; and ⑤Detection and re-verification for these strongly related genes will be necessary to make sure which key genes and strongly related genes of apoptosis, angiogenesis, and immune are involved in the gene-control pathway and mechanism during liver regeneration. Male Wistar rat around 200g will be used as subject. Partial hepatectomy 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 liver regeneration 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: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 liver regeneration. The following methods will be performed: (1) Measure the weight of remnant liver, and the mitotic index in the remnant liver.(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. (3) the genomic survey of 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) verifying 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) verifying 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)siRNA inhibition of these apoptosis related genes expressions, to evaluate if they are inhibited, and identify their signaling pathway during liver regeneration. (7) Analyzing 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 liver regeneration. (8) Figure out an apoptosis concerned gene-signaling pathway for liver regeneration after fully understanding the roles of apoptosis key genes and their strongly related genes according to the genes’ characteristics and their relationship to liver regeneration. Then we hope to confirm the apoptosis related gene-control mechanism of initiation, differention and termination during the liver regeneration. 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 liver regeneration. The following methods will be performed: (1) Measure the weight of remnant liver, and the mitotic index in the remnant liver.(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. (3) the genomic survey 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) verifying 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) verifying 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)siRNA inhibition of these angiogenesis related genes expressions, to evaluate if they are inhibited, and identify their signaling pathways during liver regeneration. (7) Analyzing 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 liver regeneration. (8) Figure out an angiogenesis concerned gene-signaling pathway for liver regeneration after fully understanding the roles of apoptosis, angiogenesis key genes and their strongly related genes according to the genes’ characteristics and their relationship to liver regeneration. Then we hope to confirm the angiogenesis related gene-control mechanism of initiation, differention and termination during the liver regeneration. 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 liver regeneration. The following methods will be performed: (1) Measure the weight of remnant liver, and the mitotic index in the remnant liver.(2) Search for the gene-control pathways which are related to the immune 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. (3) the genomic survey 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) verifying 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) verifying 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)siRNA inhibition of these immune related genes expressions, to evaluate if they are inhibited, and identify their signaling pathway during liver regeneration. (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 liver regeneration. (8) Figure out an immune concerned gene-signaling pathway for liver regeneration after fully understanding the roles of apoptosis, angiogenesis, and immune key genes and their strongly related genes according to the genes’ characteristics and their relationship to liver regeneration. Then we hope to confirm the apoptosis, angiogenesis, and immune related gene-control mechanism of initiation, differention and termination during the liver regeneration.