2012-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/659770摘要：肝臟移植往往是肝病末期和急性肝衰竭病患最後一個治療的選項，然而缺乏肝臟捐贈者一直是全球性的問題，肝臟細胞移植是一個極具潛力的替代性方案，但是肝臟細胞的來源卻更為有限；胚胎幹細胞(Embryonic stem cells)目前被認為是可以在體外無限制放大並且具有分化成包括肝臟細胞在內的高度分化能力細胞，然而這種方式還是有免疫排斥的缺點，是故個人化的胚胎幹細胞似乎更加適合用來避免免疫排反映，這種個人化胚胎幹細胞可以藉由體細胞核轉移(somatic cell nuclear transfer)技術來產生，然而這種方式並未在人類細胞上成功，最近，一種將人類纖維母細胞誘導成近似人類胚胎幹細胞-萬能細胞(induced pluripotent stem cell)-的技術被研發出來，相較於早先的方式此技術更加簡易和穩定，這技術牽涉到使用病毒(反轉錄病毒或腺病毒)載體來將3~4個全能基因(Oct4, Sox2和Klf4或Oct4, Sox2, Nanog 和Lin28)送入纖維母細胞，理論上來說，利用患者自己的皮膚纖維母細胞誘導而成的萬能細胞，將之分化成肝臟細胞進行移植就能避免免疫排斥反應，此外，建構擁有多種不同人類白血球組織抗原的萬能細胞細胞庫能夠幫助篩選到更加適合進行移植的萬能細胞株。基於安全的考量，被病毒載體操弄後的細胞並不被允許進行臨床上的移植，因此，除非萬能細胞能夠藉由非病毒和無載體的方式被製造，不然上述的夢想永遠也無法被實現。在這份計劃中，我們將嘗試利用攜帶全能性基因的合成mRNAs/miRNAs來替代原本所使用的病毒載體來誘導內皮纖維母細胞形成萬能細胞，我們將藉由檢測基因表現、畸胎瘤形成能力和體外分化能力來檢視生成之萬能細胞之全能性，並且特別專注在肝臟細胞分化之能力，而分化出來之肝臟細胞將被移植入肝毒素誘導肝臟受損之小鼠體內，植入之細胞將藉由免疫染色、螢光原位雜合技術和利用酵素免疫分析法偵測人類血清白蛋白來評估其增生能力和肝功能指標，本計畫之結果也許能夠生產出臨床上能夠使用之萬能細胞並且更進一步幫助建立一個擁有多樣人類白血球組織抗原的萬能細胞細胞庫。<br> Abstract: Liver transplantation is usually the single last choice of therapy towards end-stage liver diseases or acute liver failure. However shortage of liver organ donation is always a worldwide problem. Hepatocyte transplantation becomes a promising alternative. But sources of primary hepatocytes for transplantation are even more limited. Embryonic stem (ES) cells can be propagated unlimitedly, and can be differentiated in vitro into various types of cells including hepatocytes. But this approach may still elicit a drawback of immune rejection. Thus custom-made ES cells will be more ideal for the usage to avoid rejection. Such kind of customized ES cells may be produced by the technique of somatic cell nuclear transfer. Unfortunately such approach was still not successful in human cells. Recently a new technique was developed to induce human skin fibroblasts into ES-like pluripotent cells, the induced pluripotent stem cells (iPS). The technique is more convenient and consistent. It involved virally (either retrovirus or lenti virus) mediated delivery of 3~4 pluripotent genes (Oct4, Sox2 and Klf4 or Oct4, Sox2, Nanog and Lin28) into skin fibroblasts. Theoretically, transplantation using hepatocytes differentiated from iPS cells that are derived from patients’ own skin fibroblasts would not elicit rejection. Otherwise, setting up a cell bank containing iPS cells with various HLA phenotypes would facilitate the selection of iPS clones most suitable for transplantation into individual patients.Cells that have been manipulated with viruses would never be allowed to be used clinically for transplantation due to safety concern. Thus the dreams described above may never come true unless the iPS cells can be derived by virus- and vector-free methods. In this project, we’ll establish iPS cell clones by delivery of synthetic mRNAs/miRNAs, in substitution to virus-mediated delivery of pluripotent genes, into dermal fibroblasts. These derived iPS cells will be tested for the pluripotency by checking gene expression profiles, teratoma formation in SCID mice, and their capacities for in vitro differentiation, especially into hepatocytes. These hepatocytes will be transplanted into SCID mice pretreated by hepatotoxins for inducing liver injury. The engraftment and functioning of the human hepatocytes in the mice liver will be evaluated by immunohistochemistry, fluorescence in situ hybridization, and ELISA for human albumin in the mouse sera. The results of this project may either generate clinically useful iPS cells or further help establish the iPS cell bank harboring most HLA traits in our country.