國立臺灣大學醫學院外科袁瑞晃2006-07-262018-07-112006-07-262018-07-112003-07-31http://ntur.lib.ntu.edu.tw//handle/246246/24502在許多組織中均存在有幹細胞負責組 織的更新及修復功能，至於肝臟幹細胞的 存在問題則已經爭議多年，而目前較為人 所接受的觀念是肝臟確實存在具有幹細胞 特性的細胞，在特定的情況下此類細胞可 以被活化而分化成為成熟肝臟細胞，由於 此類細胞為不成熟的上皮細胞，具有卵圓 形的細胞核而細胞質極少因此被稱為卵圓 細胞。肝臟細胞移植研究使用的成熟肝細 胞分離後雖然可以加以培養，但數代之後 肝細胞便呈現老化現象，是目前無法克服 的障礙。由許多的研究顯示，人類病態肝 臟中也存在此類卵圓細胞，因此若能成功 的由病態肝臟中分離並培養肝臟卵圓細 胞，並實際將之移植於病態肝臟中就顯得 非常重要。本實驗的目的在於藉由化學藥 物以及大量肝臟切除的方法製造病態肝臟 以活化肝臟卵圓細胞，並將卵圓細胞由肝 臟中分離出來加以培養，再將卵圓細胞移 植到活體小鼠的病態肝臟中，以研究卵圓 細胞移植是否可以提供病態肝臟的再生。 本實驗將使用C57BL/6 以及DPPIV (Dipeptidyl Peptidase IV) knock-out 兩種不 同的小鼠，首先將C57BL/6 小鼠連續餵食 Acetylaminofluorene (AAF, 7.5mg/kg/day) 十四天，其中於第七天時進行百分之七十 的肝臟部分切除術，餵食完成之後利用 two-step collagenase perfusion 方式分離剩 餘肝臟中的卵圓細胞加以培養，同時測定 其albumin（當膽管所產生的幹細胞進入肝 臟基質中便會產生albumin）、cytokeratin 7、Thy-1（為cell surface glycoprotein 可見 於hematopoietic stem cell 及oval cell）的表 現。此外利用DPPIV 突變的小鼠，同樣利 用AAF 抑制其肝臟細胞再生能力，在進行 大量肝臟切除的同時移植由C57BL/6 分離 培養之卵圓細胞，最後在術後兩週、四週、 兩個月、四個月及六個月時犧牲取其肝臟 利用組織化學染色以區別呈現DPPIV的外 來移植細胞，並利用albumin、cytokeratin 7、Thy-1 的表現來測定移植卵圓細胞的分 化情形。Stem cells with multiple differentiation potential have been verified in several different tissues. The existence of a liver stem cell was controversial for many years, but it is now generally accepted that liver contains kinds of cells with stem-like properties and these cells can be activated under certain conditions and differentiated into hepatocytes. By reason of their oval shaped nucleus and scanty cytoplasm, these immature epithelial cells were called “oval cells”. Even though primary culture of the hepatocytes had been done for years, the 3 impacts on proliferative ability and viability after several generations made their usage limited. Under a variety of pathological conditions, similar small cells can be found in human liver too. Therefore, searching for the hepatic stem cells that can be cultured for a long period and application of these cells in hepatic cell transplantation become more and more imminent. In this study, we use chemical agent combined with partial hepatectomy as a model of diseased liver to activate hepatic oval cells for isolation and cell culture. Then these cells will be transplanted into the diseased mice in order to verify the ability of differentiation and repopulation of oval cells in vivo. Both C57BL/6 and DPPIV (Dipeptidyl Peptidase IV) knock-outmice will be used in this study. C57BL/6 mice will receive oral gavage of Acetylaminofluorene(AAF, 7.5mg/kg/day, dissolved in polyethylene glycol) for 14 days totally. Two-third partial hepatectomy will be performed at the 24 hours after the first six daily gavages (day 7th). Hepatic oval cells will be isolated using two-step collagenase perfusion method and seeded in collagen-coated tissue culture dishes. Immunohistochemical studies about the γ-glutamyl transpeptidase、 α-fetoprotein、albumin、cytokeratin 7、 cytokeratin 18、cytokeratin 19、Thy-1 will be used to evaluated the characters of these cell.. Then the oval cells will be transplanted into AAF pretreated PPIV knockout mice immediately after two-third partial hepatectomy. The recipients will be sacrificed two weeks, four weeks, two months, four months and six months after the cell transplantation. Histochemical staining will be used to distinguish the donor cells and albumin、cytokeratin 7、Thy-1 will be used to identify the character of oval cell proliferation and differentiation.application/pdf200572 bytesapplication/pdfzh-TW國立臺灣大學醫學院外科卵圓細胞分離卵圓細胞移植肝臟部分切除術cytokeratinThy-1oval cell isolationoval cell transplantationpartial hepatectomy[SDGs]SDG3reporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/24502/1/912314B002250.pdf