建立擬人化主要組織相容性複合體免疫模式小鼠

許多報導顯示，人類白血球抗原 ( human leukocyte antigen, HLA ) 基因的變異和許多免疫疾病有關連。因此建立起帶有人類 HLA 基因敲入 (knock-in, KI)的擬人化小鼠，將有助用於免疫反應疾病研究。現今雖已有許多相關小鼠模型，然而其多為「轉基因小鼠」，將基因以「隨機嵌合」的形式轉入小鼠基因組中，可能會有無法避免的副作用產生。因此，本研究旨在建立兩株擬人化的免疫小鼠模型，並分別帶有第一類以及第二類人類HLA基因，可用於我們有興趣的Graves’ disease 或是其他免疫疾病研究。同時建立導入人源的「表面抗原分化簇 4 受體 (Cluster of Differentiation 4 receptor, CD4) 」、「表面抗原分化簇 8 受體 (Cluster of Differentiation 8 receptor, CD8) 」以及「β2 微球蛋白 ( Beta 2- microglobulin, β2M )」三個在協助抗原呈現過程中重要的蛋白的小鼠模型，以確保小鼠體內免疫機制更接近人體。我們分別採用了 Crispr-Cas9 以及傳統的同源臂重組技術，將人源基因嵌入小鼠中。我們最終目標是預期能將五株小鼠以配種方式，產生帶有完整人類白血球抗原系統的擬人化小鼠模型。目前我們已獲得擬人化的 B2M 以及 CD4 小鼠。我們發現在帶有人類 B2M 基因的純合子擬人化小鼠當中，小鼠無法順利表達自身的一類主要組織相容性複合體 (major histocompatibility complex class I, MHC class I) 的 H-2Kb 分子，而另一種 MHC class I 分子 H-2Db 的表現則是異常表現。而小鼠體內的 CD4-CD8+ T 細胞比例仍與野生型小鼠類似。體外細胞培養實驗，發現擬人化的 B2M小鼠在T細胞的增生功能並未有異常現象。總體來說，這株品系小鼠雖然有上述蛋白表現異常狀況，但尚未發現有過早死亡現象發生或是其他免疫反應，不過有關免疫學上的功能仍有待後續研究。另外一株已經獲得的小鼠品系為擬人化的CD4小鼠。該品系小鼠成功表達人類的CD4基因。在細胞表面蛋白表現上，有表達人類CD4。上述表現的實驗結果也符合我們的預期。體外細胞培養實驗，也顯示出該品系小鼠T細胞增生活化能力和野生型小鼠相似。總結來說，我們並未在擬人化CD4小鼠品系上，有觀察到異常的免疫現象。而上述的觀察結果，也有助於我們後續對免疫相關的議題研究。

關鍵字

人類白血球抗原；表面抗原分化簇 4 受體； β2 微球蛋白；擬人化小鼠模型

並列摘要

Many reports have implied that the variants of human leukocyte antigen (HLA) are associated with immune-related disease. Thus, establishing a humanized HLA mouse model may have a great contribution to immunology research. Today many kinds of HLA-mouse model have been established however they still have some limitations. Most of the mouse models are “transgenic mice” which means that they will “randomly integrates” the target gene into the host genome. As a result, this study aims to establish two strains of humanized HLA mouse model which respectively carry HLA I and HLA II gene and would be helpful for immune research. In addition, we also establish another three mouse models: human CD4, human CD8 and human β2M knock-in mouse. They are the three-key molecular during the antigen presenting process. We adopt CRISPR/Cas9 and homologous recombination strategy to knock target specific gene into the mouse line. The final goal is to inbreed the five strains of the knock-in mouse and get a completely humanized HLA mouse model. Now we only have two mouse strains, humanized B2M and humanized CD4 mouse strains. We found that humanized B2M mouse do express the humanized B2M in genome DNA, mRNA and protein level on the cell surface. However, the humanizes B2M mouse strain have a relatively low expression of mouse MHC complex, H-2Kb. on the other hand, another mouse MHC complex H-2Db have abnormally high expression. The humanized B2M mouse strain have similar amount of CD4-CD8+ T cell to wild-type mouse strain. Additionally, in vitro cell culture showed that T cell proliferation ability of KI mice had no significant different compared to wildtype mice. In brief, although the humanized B2M mouse has abnormal expression of H-2Kb on cell surface, this mouse line doesn’t appear to premature die or initiate abnormal immunoreaction. However, the immune function of the humanized B2M mouse is still need the subsequence functional assay to test. Another mouse strain we have acquired is hCD4 mice. The hCD4 KI mice indeed expressed human CD4 in gDNA level. It also expressed equal mCD4 and hCD4 on cell surface. This result lined with our expectations. In vitro cell culture experiment analysis also depicted that T cell proliferation ability were similar to wildtype mice. In summary we didn’t observe abnormal immune phenomenon from the CD4 mouse strain. The above result will be helpful to immune-related research in the future.

並列關鍵字

Human Leukocyte Antigen (HLA) ； Cluster of Differentiation 4 receptors (CD4) ； Beta 2-Microglobulin (B2M) ； Humanized Mouse Model

參考文獻

1. Wieczorek, M., et al., Major histocompatibility complex (MHC) class I and MHC class II proteins: conformational plasticity in antigen presentation. 2017. 8: p. 292.

Google Scholar

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