https://scholars.lib.ntu.edu.tw/handle/123456789/192307
標題: | 以基因微陣列分析肝細胞再生時,特種基因及基因族群之基因表現在程度、型態、時程的變遷並鑑別其扮演之角色 ANALYZING THE REGULATING GENES OF LIVER REGENERATION IN CHANGING DEGREE, PATTERN, TIMING AND VERIFYING THE ROLES OF SPECIFIC AND CLUSTER GENES BY cDNA MICROARRY |
作者: | 賴鴻緒 | 關鍵字: | liver regeneration;partial hepatectomy;proto-oncogene;microarray;genetic changing pattern;gene cluster;肝細胞再生;部分肝臟切除術;基因微陣列;基因表現型 態;基因族群 | 公開日期: | 31-七月-2005 | 出版社: | 臺北市:國立臺灣大學醫學院外科 | 摘要: | 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 基因控制之詳細機轉,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、p21、gas-6 及ras 家族基因等的表現 的確有增加現象。Arora 等人發現抗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 等之表現也曾被提出與肝細 胞再生有關,但大量搜尋相關基因變化之 研究,則尚未有報告。吾人初步以384 點 肝臟相關之cDNA基因微陣列(microarray) 研究,發現肝細胞再生過程中,有59 種基 因表現明顯增加,19 種明顯降低,但因其 變化程度、型態、時程與基因種別、基因 族群均十分複雜,無法清楚分析,其扮演 之角色也尚難了解。更大量的cDNA 基因 微陣列搜尋,以進一步分析及了解基因管 控之機轉仍屬必要。 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,超過 6000 種確定基因種別之基因表現,並分析 其變遷程度、型態、時程及各基因族群之 相關性,以了解其在肝細胞再生中確實扮 演之角色。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、12、24、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(6144 identified cDNA clones, Wittech Co, Taipei, Taiwan)、肝細胞mRNA 標號、hybridization 及影像分析等方法,測 定6144 種基因表現之變遷程度、型態及時 程;(4)將基因表現變化大者,依特性分為 免疫、賀爾蒙、生長因素、酵素、及血管 新生因子等基因族群,並比較其程度、型 態及時程之差異性。結果發現:(1)剩餘肝 臟重量比值,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)所有基因表現變遷之型態及時程共分為72 種,包括 第2、、6、12、24、72 小時及、7 天單一 尖峰型、雙尖峰、遞增型、遞減型、突出 型、凹陷型和混和型等,每種型態包括40 至218 種基因;(4)包括免疫、賀爾蒙、生 長因子、酵素及血管新生因子等基因族 群,均有明顯的變遷;(5) fas-associating protein with death domain, carnitine palmitoyltransferase 1, fas death domain-associating protein, 及steroid O-acyltransferase 1 等早期變化基因可能與 肝再生之啟動有關;(6) transforming growth factor beta 2 及beta receptor 等中期變化基 因可能與肝再生之分化有關;(7) TGF-β regulated gene 3 及small inducible cytokine A2 等晚期變化基因可能與肝再生之終止 有關。 Although there are much controversy 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. However, the regenerative mechanism and genetic control of liver after major tissue loss is still not clear. The regenerating liver is a system in which the relationships between proto-oncogene expression and cell replication should be examined during a physiologic growth response. Proto-oncogene expression after partial hepatectomy should be 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, p21, gas-6 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. Arora et al reported that c-Myc antisense limits rat liver regeneration by regulating cytochrome p-450 3A activity. Ozeki and Tsukamoto found that retinoic acid can repress c-fos and c-jun expression and induce apoptosis in regenerating liver. Our previous study monitored the variation of regulating genes by 384 liver-related gene cDNA microarray nylon membrane, and found that there are 59 proto-oncogenes expression increased markedly and 19 decreased significantly during liver regeneration. However, the changing degree, patterns, timing and gene grouping were very sophisticated and not clear. Mass survey and more detailed analysis by more cDNA microarry method should be very important. Male Wistar rats around 200g will be used as subject. Partial hepatectomy around 70% were performed. They were sacrificed before and 2, 4, 6, 12, 24, 72 hours and 5, 7, 10 days after hepatectomy. We have measured: (1)weight of remnant liver; (2)mitotic index; (3)genomic survey of the gene expression by microarray of 6144 identified cDNA clones on nylon membrane (Wittech Co., Taipei, Taiwan), labeling of liver mRNA hybridization and image analysis; and (4)Grouping of genes expression into immune, nutrition, hormone, growth factor, enzyme, oncologic and embryonic subgroups, and compare the expression degree, changing pattern and specific timing. The results were: (1) the remnant liver weight increased to 90% in 72h after partial hepatectomy; (2) the mitosis of hepatocytes increased marked at 48h then decreased at 72 after partial hepatectomy; (3) analyzing the gene expression of microarray chips, the variation could be classified into 72 different patterns in cluding the patterns with a single peak at 2, 4, 6, 12, 24, 72h and 5, 7d after partial hepatectomy; (4) gene clusters of immune, hormone, growth factor, enzyme and angiogenesis have changed markedly; (5) early stage changed genes including fas-associating protein with death domain, carnitine palmitoyltransferase 1, fas death domain-associating protein, and steroid O-acyltransferase 1 could be related to the initiation of liver regeneration; (6) intermediate stage changed genes including transforming growth factor beta 2 and beta receptor could be related to the differentiation of liver regeneration; (7) late stage changed genes including TGF-β regulated gene 3 and small inducible cytokine A2 could be related to the termination of liver regeneration. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/24534 | 其他識別: | 922314B002276 | Rights: | 國立臺灣大學醫學院外科 |
顯示於: | 醫學系 |
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