建立表現顆粒溶解素之移植物抗宿主症之免疫系統擬人化小鼠

Abstract

儘管捐贈者的毒殺型T淋巴細胞 (CTLs) 與自然殺手細胞 (NK cells) 在引起移植物抗宿主症 (graft-versus-host disease) 的角色被確立，然而對於導致病患皮膚、黏膜細胞壞死脫落及多重器官衰竭的細胞毒性蛋白的身分仍不清楚。顆粒溶解素(granulysin) 則是在最近研究中指出可能引致移植物抗宿主症的一種細胞毒性蛋白。由於小鼠的基因組中並沒有顆粒溶解素的基因，所以傳統的小鼠模式並不適合用來進一步研究顆粒溶解素與移植物抗宿主症的關係。本篇論文中，我們報告利用人類臍帶血單核細胞植入至經放射線處理過的免疫系統嚴重缺陷小鼠( NOD/Lt-scid IL2rγ null mice; NSG mice)，在免疫系統擬人化小鼠中建立異體移植物抗宿主症動物模式 (HIS-X-GVHD)。在此HIS-X-GVHD模式中，我們觀察到小鼠產生體重下降、皮膚及黏膜細胞壞死等移植物抗宿主症之症狀，並於小鼠的肝、脾、肺、腎、皮膚及血清中發現高量人類來源之免疫細胞及人類的細胞激素。免疫化學染色可見人類CD45+、CD8+之免疫細胞及顆粒溶解素在小鼠肝、脾、肺、腎及皮膚組織中高度表現。最重要的是，血清中顆粒溶解素的濃度與異種移植物抗宿主症之嚴重程度有顯著相關。 綜合以上結果顯示，此移植物抗宿主症之免疫系統擬人化之小鼠模式可提供作為顆粒溶解素作用機制的研究，並提供作為新穎治療藥物臨床前評估的新工具。

Although the involvement of cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells) in the pathogenesis of graft-versus-host-disease (GVHD) have been well established, the cytotoxic proteins that are responsible for the skin and organ toxicity remains to be clarified. Recently a mechanism involving a cytotoxic protein, granulysin, was proposed. However, study on the functional role of granulysin in GVHD has been hampered by lack of appropriate mouse GVHD model due to the absence of granulysin gene in the mouse genome. Here, we developed a humanized immune system (HIS) mouse model with xenogenic-GVHD based on the intravenous injection of human umbilical cord blood mononuclear cells (UCBMCs) into sublethally irradiated NOD/Lt-scid IL2rγ null (NSG) mice. The HIS-X-GVHD mouse model exhibited human cells engraftment and human cytokine production with lymphocytic infiltration in the liver, spleen, lung, kidney and skin. Futhermore, immunohistochemistry demonstrated that the infiltrative lymphocytes in various organs were predominantly human CD45+ and CD8+ cells. Importantly, we found that granulysin was highly expressed in the liver, spleen, lung, kidney and skin. Besides, Serum granulysin levels were positively correlated with severity of GVHD. The HIS mouse model with X-GVHD provides the opportunity to investigate in vivo mechanisms of the regulation of granulysin, as well as to test the novel therapeutics for treatment of GVHD.