2018-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/702376摘要：除了靈長類之外，豬隻器官被認為是解決人類器官來源短缺的最佳替代品。但是以一般豬隻的器官進行人類異種移植，將遭遇複雜之排斥反應，包含1.補體與自然抗體所引起之超急性排斥；2.血管性及細胞性之急性排斥；與3.慢性排斥。此外，異種抗原Non-Gal，亦可能在異種移植排斥扮演著重要角色。急性細胞性排斥反應是移植後，豬隻器官抗原活化人類 T-細胞與人類自然殺手(nature killer, NK)細胞，而後破壞植入之豬隻器官。基因改造之豬隻器官，可以克服部分之異種移植排斥反應，目前主要是以克服超急性排斥為目的。目前我國財團法人農業科技研究院-動物科技研究所 (ATIT) 已有 CD55/hHO-1 雙基因轉殖豬、GGTA1/CMAH KO 雙基因剔除豬，可供研究。CMP-N-acetylneuraminic acid (CMP-Neu5Ac；NANA) 分子與N-Glycolylneuraminic acid (Neu5Gc；NGNA) 分子，是非人類哺乳類細胞所有之 sialic acid 分子，控制細胞Neu5Gc分子製造過程的關鍵基因是CMAH基因，人類細胞沒有CMAH基因，所以人類細胞只有CMP-Neu5Ac (NANA)分子，而豬隻細胞同時有Neu5Ac (NANA) 分子與 Neu5Gc (NGNA)分子；CMAH雙基因剔除豬細胞只有Neu5Ac (NANA) 分子，與人類細胞相似。理論上，CMAH雙基因剔除豬細胞比較不易被人類組織排斥掉，而普通豬細胞比較容易被人類組織排斥掉。脂肪幹細胞 (adipose-derived stem cell, ADSC)，可經由分化及免疫調節作用，促進移植體之存活，使其近來成為移植研究重點。由於脂肪幹細胞具有上述能力，加上取脂肪組織分離細胞的風險低，脂肪幹細胞遂成為一良好的細胞治療工具。本研究計畫之目的，乃是利用臺灣動物科技研究所(ATIT)產製之CMAH雙基因剔除豬，以一年時間進行異種移植之動物實驗，探討脂肪幹細胞減緩CMAH雙基因剔除豬異種皮膚移植之急性細胞性排斥之程度。選擇hPBMC-ASID小鼠-異種皮膚移植的實驗模式。本計畫為1年期計畫。所有基因剔除豬均以PCR、IHC stain進行分析，並測量豬隻皮膚異種移植排斥時間，與病理切片分析(H&E stain，IHC stain for CD3、CD4、CD8)。<br> Abstract: Starting from early 2000, with the introduction of galactose-α1,3-galactose (Gal)-knockout pigs, prolonged survival especially in heart and kidney xenotransplantation was recorded. However, remaining antibody barriers to non-Gal antigens continue to be the hurdle to overcome. The production of genetically engineered pigs was difficult requiring prolonged time. However, advances in gene editing, such as zinc finger nucleases, transcription activator-like effector nucleases, and most recently clustered regularly interspaced short palindromic repeats (CRISPR) technology made the production of genetically engineered pigs easier and available to more researchers. Today, the survival of pig-to-nonhuman primate heterotopic heart, kidney, and islet xenotransplantation reached more than 900, more than 400, and more than 600 days, respectively. The availability of multiple-gene pigs (five or six genetic modifications) and/or newer co-stimulation blockade agents significantly contributed to this success. Now, the field is getting ready for clinical trials with an international consensus. The Animal technology institute Taiwan of Agricultural Technology Research Institute had produced CD55/hHO-1 double transgenic pigs and GGTA1/CMAH KO double knockout pigs available for research.Humans cannot synthesize N-Glycolylneuraminic acid (Neu5Gc) because the human gene CMAH is irreversibly mutated, though it is found in apes. It is absent in human tissues because of inactivation of gene encoding CMP-N-acetylneuraminic acid hydroxylase. The gene CMAH encodes for CMP-N-acetylneuraminic acid hydroxylase, which is the enzyme responsible for CMP-Neu5Gc from CMP-N-acetylneuraminic (CMP-Neu5Ac) acid. Neu5Gc is closely related to the commonly known N-acetylneuraminic acid (Neu5Ac). Neu5Ac and Neu5Gc molecules are transferred into the Golgi so that they may be added to many glycoconjugates. However, in humans, Neu5Gc is not present. In the CMAH KO double knockout pigs, Neu5Gc is not present either. So, the human tissue might not rejection the cells /or tissue from the CMAH KO double knockout pigsLipoaspirate, the waste product of liposuction, contains a large population of regenerative cells called adipose derived regenerative cells (ADRCs), which share a number of similarities with bone marrow cells, including the capacity for multilineage differentiation. During the past decade, numerous studies have provided preclinical data on the safety and efficacy of adipose-derived stem cells, supporting the use of these cells in future clinical applications. Various clinical, including immunomodulation in allotransplantation. In this study, we will transfer the skin of CMAH KO double knockout pigs to the back of hPBMC-ASID mouse, and test the attenuation effects of adipose derived regenerative cells (ADRCs) which will be injected to the hPBMC-ASID mouse (IP) for immunomodulation.異種移植異種皮膚移植脂肪幹細胞CMAH雙基因剔除豬急性細胞性排斥XenotransplantationSkin XenotransplantationAdipose-Derived Stem CellAcute Cell-mediated RejectionCMAH Double KO PigletHumanized ASID Mice