https://scholars.lib.ntu.edu.tw/handle/123456789/192276
標題: | 以基因微陣列監測大鼠肝臟部分切除後, 肝細胞再生相關基因之變遷 MONITORING THE VARIATION OF REGULATING GENES BY c-DNA MICROARRAY DURING LIVER REGENERATION AFTER PARTIAL HEPATECTOMY IN RATS |
作者: | 賴鴻緒 | 關鍵字: | 肝細胞再生;部分肝臟切除術;proto-oncogene;基因微陣列;雜交;liver regeneration;partial hepatectomy;microarray;hybridization | 公開日期: | 31-七月-2003 | 出版社: | 臺北市:國立臺灣大學醫學院外科 | 摘要: | 肝臟是人體最重要的維生器官之 一,當肝細胞大量損傷或切除時,可因肝 衰竭導致死亡。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 證實多種營養素、賀爾蒙、生長因子、藥 劑等,可直接或間接影響肝細胞再生,但 肝細胞再生之詳細機轉及基因控制過程, 則仍不明瞭。 任何細胞之分裂與再生,必然與細胞 核內proto-oncogene 之表現有相當大的關 聯,肝細胞再生也不例外。在多項以mRNA 定量的研究報告中指出,肝臟經部分切除 後,剩餘肝臟內某些基因如(c-fos、c-myc、 p53 及ras 家族基因等)的表現的確有增加 現象。Arora 等人則發現抗c-myc 物質可經 由p-450 3A 活性之調控可抑制肝細胞再 生;Ozeki 及Tsukamoto 發現retinoic acid 可抑制c-fos 及c-jun 之表現,促成肝細胞 凋亡而使再生程度減少;而其他基因如p21 及gas-6 等之表現也被提出與肝細胞再生 有關,但以基因微陣列(microarray)來大 量監控肝細胞再生過程中相關基因之變 遷,則仍未有報告。 本計劃以重約200 克之Wistar 雄性大 鼠做實驗,測定肝細胞再生過程中,多種 基因表現在各時段之變遷。所有大鼠均接 受約百分之七十之肝臟部分切除手術,各 於術前及術後2、4、6、8、10、12、24、 48、72 小時及5、7、10 天後犧牲取樣,測 定(1)剩餘肝臟之重量比值;(2)剩餘肝臟之 有絲分裂指標;(3)以基因微陣列尼龍膜、 肝細胞mRNA 標號、hybridization 及影像分 析等方法,測定超過300 種基因表現之變 遷。結果發現:(1)剩餘肝臟之重量於切肝 術後72 小時即恢復90%以上;(2)有絲分 裂於術後48 小時大量出現,術後72 小時 逐漸減少;(3)肝細胞再生過程中,基因表 現之變化共可分為72 種型態,包括單一尖 峰、雙尖峰、遞增型、遞減型、突出型、 凹陷型或混和型等,每種型態包括40 至 218 種基因。以基因微陣列大量監測分析肝 細胞再生相關基因的變化,在研究肝細胞 再生基因機轉方面,的確扮演相當重要的 角色。 Liver is a core organ in our body. Although it contains good regeneration ability, hepatic failure can still occur after massive hepatic injury or hepatectomy. Because of the high incidence of hepatitis B carrier and so as to many hepatoma patients, the mortality rate of hepatic disease (including hepatoma cases) is one of the tope ten causes of death in Taiwan. Although there are much controversy continues on the initiation, regulation, metabolic changes, and termination of liver regeneration after partial hepatectomy that well initiate proliferation of the remaining hepatocytes, several factors, such as hormones, growth factors, nutritional components, and pharmacological agents, have been demonstrated to directly or indirectly affect liver regeneration. The regenerating liver is a system in which the relationships between protooncogene expression and cell replication should be examined during a physiologic growth response. Proto- oncogene expression after partial hepatectomy is specific, sequential, and highly regulated. As measured by levels of mRNAs, the changes have been detected in the expression of c-fos, c-myc, p53, and the ras gene family (c-Ha-ras, c-Ki-ras, and N-ras). In contrast, expression of c-src and c-abl does not change after partial hepatectomy while c-mos transcripts cannot be detected in normal or regenerating liver. In recent study, the critical roles of some proto-oncogenes were noted in the control of cell proliferation, differentiation, and apoptosis by the new technique of complementary DNA microarray. However, mass survey and more detailed analysis by microarry method, that will be very important, are still nor reported. The main purpose of this project is to find out the variation of more than 300 regulating genes during liver regeneration after partial hepatectomy. Male Wistar rats around 200g were used as subject. Partial hepatectomy (around 70%) were performed and they will be sacrificed at 2, 4, 6, 8, 10, 12, 24, 48, 72 hours and 5, 7, 10 days after hepatectomy. We measured: (1) weight of remnant liver; (2) mitotic index; and (3) genomic survey of the expression for more than 300 proto-oncogenes by cDNA microarray on nylon membrane, labeling of liver mRNA hybridization and image analysis. We found that: (1) the remnant liver weight increased to 90% in 72h after partial hepatectomy; (2) the mitosis of hepatocytes increased markedly at 48h then decreased at 72h after partial hepatectomy; (3) analyzing the gene expression of microarray chips, the variations could be classified into 72 different patterns including the pattern with a single peak at 2, 4, 6, 12, 48, 72h and 5, 7 days time sequence after partial hepatectomy. The variation patterns also included double peaks, enhancing trend, diminished trend, protruding curve, excavated curve and other mixed types. Each category of gene expression pattern contained 40 to 218 proto-oncogenes. The quantitative gene expression profiles should have important implications for the investigation on the mechanism of regenerating process. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/24501 | 其他識別: | 912314B002247 | Rights: | 國立臺灣大學醫學院外科 |
顯示於: | 醫學系 |
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912314B002247.pdf | 2.18 MB | Adobe PDF | 檢視/開啟 |
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