2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/645386摘要：肝細胞移植已被證實可以用於治療許多的肝臟疾病，但這樣的治療方式受到了許多的限制，主要包含異體移植後的排斥問題、肝細胞的捐贈來源有限、移植須要大量的肝細胞以及肝細胞無法於體外進行培養增殖後再進行移植。近來的研究證實藉由轉殖4個轉錄因子(Oct4、Klf4、Sox2與Myc)進入已成熟的體細胞內(如皮膚纖維母細胞)可以誘使細胞重整並且產生去分化現象進而生成引導式多能性幹細胞。此外，藉由轉殖細胞系特有轉錄因子亦可促使成熟細胞轉分化為其他系別的細胞。因此，利用病人本身細胞來產生移植所需的細胞將可直接解決細胞來源與之後免疫排斥的問題，對於臨床治療上將有極大的應用潛力。最近，小鼠的皮膚纖維母細胞已經被證實可轉分化為具有肝細胞功能的引導式類肝細胞，而這樣的過程只需要轉殖2(HNF-4α與Foxa1、Foxa2或Foxa3)到3個(Gata4、HNF1α與Foxa3)不同肝臟發育相關的轉錄因子即可達成，但是這樣的研究結果目前仍無法應用於人類皮膚纖維母細胞上。在這個研究計畫中，我們的首要目標是於體外產生人類誘導類肝細胞。其方式為利用慢病毒轉殖多種肝臟發育相關的轉錄因子到人類皮膚纖維母細胞中以誘使該細胞進行轉分化而生成類肝細胞，並藉由檢測基因表現、免疫螢光染色以及肝細胞功能分析等方式來評估所產生的類肝細胞與正常肝細胞的相似程度。此外，生成的類肝細胞也會與其他子計畫的研究相結合，應用於藥物性肝損傷的NOD/SCID小鼠細胞移殖治療實驗，並以組織螢光免疫染色及血清生化分析等方式驗證類肝細胞是否有發育為成熟且具有功能的肝細胞能力。另外，我們也會分析各類肝細胞株內部外源轉錄因子基因表現組合情況，以找出誘使類肝細胞生成所需的最適當轉錄因子組合。而本研究計畫的另一目標是建立一無病毒、無載體的誘導類肝細胞生成方式。以體外合成各轉錄因子mRNA取代病毒或是載體送入皮膚纖維母細胞內以促使細胞轉分化產生誘導肝細胞，這樣的方法建立將有助於提高日後使用在臨床治療上的安全性。本研究之結果，將有助於為日後肝臟疾病的治療提供一穩定且病人專有的肝細胞來源。<br> Abstract: Hepatocyte transplantation is an option and has been proven effective for patients with acute liver failure and certain metabolic diseases. However, this application for clinical usage has several obstacles including immunological rejection, limited sources of hepatocytes, large number of cells needed for transplantation and loss of normal hepatocyte function if in vitro culture expansion was attempted. Recently, several evidences showed that transduction of four transcription factors (Oct4, Klf4, Sox2 and Myc) can induce somatic cells (e.g skin fibroblasts) “reprogrammed” to induced pluripotent stem cells that also call “induced pluripotent stem cells”. Moreover, several studies have shown that overexpression of linage-specific transcription factors could directly convert terminally differentiated cells to other lineages, a process called “cell transdifferentiation”. For example, direct conversion of mouse fibroblasts into hepatocyte-like cells has been accomplished by transduction of three (Gata4, HNF1α and Foxa3) or two (HNF-4α plus Foxa1, Foxa2 or Foxa3) liver development-related transcription factors. However, it is still unknown whether this approach may be applied to human fibroblasts.In this subproject, we’ll try to convert human dermal fibroblasts into induced hepatocyte-like cells by lentiviral transduction of several liver development-related transcription factors into fibroblasts. The induced hepatocyte-like cells will be evaluated by gene expression profile, immunohistochemistry staining and in vitro hepatocyte functional assays. Moreover, we will study the in vivo repopulation ability and function of induced hepatocyte-like cells by transplantation into liver-injured NOD/SCID mice and evaluate by immunohistochemistry staining and biochemical analyses. Furthermore, based on the results indicating optimal transcription factors needed to transdifferentiate dermal fibroblasts, we’ll then establish a method of producing virus- and vector-free human induced hepatocyte-like cells by mRNA-transfection technique. The result of this study will help us to develop the novel source of patient-specific hepatocytes for clinical hepatocyte transplantation.