異種器官供人體移植之可行性研究-使用多基因轉殖小鼠與豬器官─HO-1/hDAF/HLA-DR多基因轉殖豬細胞體外之功能測試(1/2)

以豬器官來源的異種器官，為目前臨床移植所面臨的器官短缺的問題，提供 了解決之道。但是其可行性，目前仍受限於人類對豬隻的異種抗體所誘發的超急 性，及亞急性異種器官排斥，及物種間組織抗原歧異所引起的T 細胞排斥。先前 在我們所產製的HLA DPW0401 基因轉殖豬中，發現人類HLA DP 的抗原性，可完 整表達在豬細胞表面，而相較於其他非基因轉殖同胞豬，HLA DPW0401 基因轉殖 豬所引起人類周邊單核球(具HLA DPW0401 基因型者)的增生反應，有減少的趨勢。 HO-1 是代謝Heme 的主要酵素，而能保護細胞抵抗異種抗體所引發的細胞裂 解及凋亡,及使異種移植器官在異種抗體存在之下，達到”適存化”的狀態。而 人類延遲加速因子(hDAF, CD55)為一補體活化鏈的調節者。目前發現 hDAF 的基 因轉殖豬器官，包括心臟，腎臟及肝臟可在靈長類體內， 存活7 天至3 個月。 在過去一年中，我們已經成功地產製了HLA-DR 及HO-1 之基因轉殖豬，其 mRNA 及其蛋白的表達都已得到確定，針對HO-1 保護作用的測試，我們已經成功 地完成自然殺手細胞毒性測試的檢驗模式，我們發現殺手細胞與標的細胞於5： 1 的情形有最明顯的毒殺現象，之後我們將運用於HO-1 轉殖豬細胞的測試，檢 測此HO-1 轉殖基因是否有保護作用。我們也完成了HLA-DR 基因轉殖豬細胞對人 類週邊單核球的增生測試，我們發覺HLA-DR 轉殖基因有減緩人類周邊單核球異 種細胞反應的趨勢。至於這些抑制作用是透過直接或間接的抗原抗體認識，而產 生作用，目前仍進一步確認中。由於異種移植所引起的免疫反應廣泛地涉及細胞 及體液免疫反應，因此多基因轉殖以克服不同層面的問題，及有其必要性，因此 針對不同基因的特定功能測試是前臨床試驗的重要步驟。Pig to human xenotransplatation is a promising strategy to overcome the organ shortage in clinical transplantation. However, the application is hampered by the xenoreactive antibody mediated hyperacute rejection, delayed xenograft rejection and MHC disparity associated T cell rejection. Previous, we have produced the HLA DPW0401 transgenic pig, which was shown equipped with the human HLA antigenecity and, as compared to the non-transgenic litermate pig, induced a minor cellular response to the human PBMCs that came from the HLA DPW 0401(+) human. HO-1 (heme oxygenase-1) is the main enzyme metabolizing the heme, which has been demonstrated to offer a protective effect for the xenograft to achieve accommodation under the presence of xenoreactive antibodies, and help to attenuate xenoantibody mediated celll lysis and apoptosis. To test the protective effect of HO-1 exogene from the NK related cytotoxicity, we have establish the optimal condition for this cytotoxic assay using the NK cell line, K562. The cell killing efficacy will be maximal when the effector/target ratio being 5: 1, 47% of cell death of the total cell population. We have also successfully cultured the aortic endothelial cells of pig (PAEC) from the non-transgenic pig. The culture of the PAEC of the transgenic pig is ongoing now. Because the HLA-DR 1501 has been successfully produced, we also test the xenogenic MLC for the HLA DR transgenic pig. We found the HLA DR exogene once expressed on the pig cell has a tendency to reduce the xenogenic cellular response if the human responder PBMC has the same HLA DR gene as the transgenic pig. The confirmative study is on the way at the present time