擬人化CD8小鼠以及擬人化CD4小鼠之表徵分析

Abstract

葛瑞夫茲氏病（Graves' disease, GD）為甲狀腺機能亢進之疾病之一，其亦為常見之自體免疫甲狀腺疾病，而人類白血球抗原（human leukocyte antigen, HLA）基因之多態性（polymorphism）與GD之引發相關，兩者之關聯仍待釐清。前人研究 HLA之動物模式較常以轉基因小鼠為主，但其以隨機性基因插入方式，具非特異性位點插入之問題，影響致病機制之表現，而本研究則利用CRISPR-Cas9技術及其特性，精確且特異性敲入目標基因，建立擬人化CD4（humanized CD4 knock in mice, hCD4 KI mice）及CD8（humanized CD8 knock in mice, hCD8 KI mice）兩種協助HLA抗原呈獻基因之小鼠模式，以利探討GD以及HLA相關之疾病。本實驗室前人已成功建立hCD4 KI mice及hCD8 KI mice模式，但其DNA、蛋白質及功能性仍未探討，故本研究旨在鑑定與分析兩小鼠模式之DNA，以及其T細胞之蛋白質表現、活化與增殖能力。依結果顯示，在DNA及蛋白質表現上，hCD4 KI mice及hCD8 KI mice分別成功攜帶human CD4（hCD4）與human CD8（hCD8）序列，淋巴細胞表面則亦分別表達hCD4和hCD8之標誌物，然從純合子hCD4 KI mice及純合子hCD8 KI mice中發現，前者胸腺中hCD4單陽性T細胞數量顯著減少，而mouse CD8（mCD8）單陽性T細胞增多，導致周邊以及脾臟成熟之hCD4+ T細胞減少、mCD8+ T細胞增多，引起CD4/CD8比值降低；後者胸腺中mouse CD4（mCD4）單陽性T細胞無顯著變化，但hCD8單陽性T細胞數量顯著減少，而使周邊成熟之hCD8+ T細胞及mouse CD3+ T細胞數量亦隨之下降，造成CD4/CD8之比值上升。不過在體重、T細胞活化與增殖、組織染色及全血液分析結果中，皆顯示兩小鼠模式與野生型小鼠無顯著差異，且實際觀察中，亦無異常死亡或重大免疫缺陷。綜合上述結果，本研究推測hCD4或hCD8之敲入對小鼠並無重大之影響，hCD4 KI mice及hCD8 KI mice可應用於未來免疫相關之研究。

Graves’ disease (GD) is one of the hyperthyroidism diseases which commonly occur as autoimmune thyroid disease, and have been shown that the cause of GD is related to the polymorphism of the human leukocyte antigen (HLA) gene. To understand the pathogenic mechanism between GD and HLA gene, the HLA transgenic mice has been established through the random insertion of transgenes in previous studies. However, this animal transgenic method is limited by the occurrence of non-specific loci insertion that may affect the pathogenic mechanism. In this study, CRISPR-Cas9 technology was applied to specifically knock in the target gene to establish the mice models. CD4 and CD8 assist in HLA antigen presentation are applied to establish the humanized CD4 knock-in mice (hCD4 KI mice) and humanized CD8 knock-in mice (hCD8 KI mice), so as to facilitate the investigation of GD and HLA related diseases. hCD4 KI mice and hCD8 KI mice are successfully established by predecessors in our laboratory, but the DNA, protein, and functionality of these two mice have not yet been explored. Therefore, this study aims to identify the DNA, and further investigate the protein expression and functionality of T cell of these two mice models. According to our results, hCD4 KI mice and hCD8 KI mice successfully carried human CD4 (hCD4) and human CD8 (hCD8) DNA sequences respectively. And, the lymphocyte surface also expressed markers of hCD4 and hCD8, respectively. In homozygous hCD4 KI mice, the number of hCD4 single positive T cells in the thymus was significantly decreased, but the number of mice CD8 (mCD8) single positive T cells in the thymus was significantly increased, which led to the decrease of hCD4+ T cells and the increase of mCD8+ T cells in peripheral blood and spleen, resulting in the decrease of CD4/CD8 ratio. In homozygous hCD8 KI mice, there was no significant change in mice CD4 (mCD4) single positive T cells in the thymus, but the number of hCD8 single positive T cells was significantly decreased in the thymus. In peripheral, the number of hCD8+ T cells and mice CD3+ T cells was decreased and resulting in an increase in the ratio of CD4/CD8. Finally, the body weight, activation and proliferation of T cell, tissue staining and whole blood analysis were no significantly difference between two mice model and wild type mice. In addition, there was no abnormal death or major immune deficiency in actual observation. Above all, this study suggests that the knock in of hCD4 or hCD8 had no significant effect on mice, and that hCD4 KI mice and hCD8 KI mice could be used in future immune related studies.