奈米金在卵白蛋白免疫小鼠中增強抗原特異性IgG2a的生成以及誘導骨髓衍生抑制型細胞

Abstract

由於奈米金 (AuNPs) 具有良好的生物相容性及抗腫瘤活性，使其被廣泛應用於藥物傳遞與癌症免疫治療。先前的研究證實奈米金經由靜脈注射後，主要累積於脾臟中並會誘導發炎細胞激素的生成。此外，奈米金分布於脾臟的免疫細胞內，包括B細胞、T細胞和骨髓衍生抑制型細胞 (MDSC)。特別是骨髓衍生抑制型細胞是攝取奈米金的重要細胞之一。儘管已知單核吞噬細胞系統 (MPS) 在清除奈米金的過程中扮演關鍵角色，奈米金對免疫細胞可能具有潛在的影響，然而奈米金與免疫系統的相互作用尚未徹底了解。因此，本論文主要研究奈米金對於卵白蛋白 (ovalbumin ; OVA) 免疫小鼠抗原專一性免疫反應之影響，以及奈米金誘發之骨髓衍生抑制型細胞於遲發性過敏反應 (DTH) 小鼠中之功能性，並且探討奈米金粒徑大小對免疫反應之影響。實驗分為三大部分，第一部分：BALB/c小鼠於第1至12天，每天以靜脈注射方式投予奈米金 (0.4 mg/kg) 或磷酸鹽緩衝生理食鹽水 (PBS) 作為對照組，在試驗第2天及第12天腹腔注射OVA進行免疫，在第13天犧牲並採集各組別小鼠之血清與脾臟進行後續實驗。實驗結果顯示，奈米金顯著增加了脾臟指數 (spleen index) 及脾臟中CD11b+Gr-1+細胞族群 (MDSC之細胞表面標記)。奈米金並沒有影響脾臟細胞之代謝活性，但促進脂多醣 (LPS) 刺激之脾臟細胞生成腫瘤壞死因子-α (TNF-α) 與介白素-10 (IL-10)，以及刀豆蛋白A (ConA) 或OVA刺激之脾臟細胞生成干擾素-γ (IFN-γ)，並且增強OVA免疫小鼠抗原專一性之抗體IgG2a。第二部分：小鼠分別於第1天及第9天經由腹腔注射與後腳掌皮下注射OVA誘導遲發性過敏反應，並在第9天進行皮下注射前尾靜脈注射奈米金組別小鼠之脾臟細胞，測量其腳掌腫脹程度。其結果表明，接受奈米金小鼠脾臟細胞之小鼠組別顯著降低腳掌腫脹程度。第三部分：小鼠於第1至8天，每天靜脈注射不同粒徑大小 (10 nm, 50 nm, 100 nm) 之奈米金 (0.5 mg/kg) 或0.1 mM生理食鹽水 (PBS) 作為對照組，於第9天犧牲。在不同尺寸之奈米金組別中，10 nm與50 nm奈米金皆增加了脾臟指數，50 nm奈米金使脾臟中MDSC細胞族群比例上升，但是不同粒徑大小奈米金所產生之發炎細胞激素表現並不具尺寸相關性。整體來說，本研究結果證實奈米金會增強抗原專一性免疫反應及第一型輔助T細胞(Th1) 之免疫，並且誘導脾臟中具有抑制活性之骨髓衍生抑制型細胞。

Gold nanoparticles (AuNPs) have been widely used for drug delivery and cancer immunotherapy due to their biocompatibility as well as antineoplastic activity. Previous studies demonstrated that systemic exposure to AuNPs mainly accumulated in the spleen and induced the production of inflammatory cytokines. In addition, AuNPs are distributed widely across the immune cells in splenocytes, such as B cells, T cells, and myeloid-derived suppressor cells (MDSC). In particular, MDSC is one of the important cells uptake AuNPs. Although the mononuclear phagocytic system (MPS) is known to play a critical role in the clearance of AuNPs, the interaction of AuNPs with the immune system has not been studied thoroughly. Therefore, my study aimed to investigate the effects of AuNPs on antigen-specific immune responses in ovalbumin (OVA)-sensitized mice and the functionality of AuNP-induced MDSC in delayed-type hypersensitivity (DTH) mice. Furthermore, the particle size influence of AuNPs on the immune response was explored. Results of this study showed that AuNPs significantly increased the spleen index and the population of splenic CD11b+Gr1+ cells. AuNPs enhanced the production of TNF-α and IL-10 by splenocytes stimulated with lipopolysaccharide (LPS) and the production of IFN-γ by splenocytes stimulated with concanavalin A (ConA) or OVA. The serum level of OVA-specific IgG2a was augmented in AuNP-treated mice. The thickness of footpad swelling in DTH mice received splenocytes from AuNP-treated mice was significantly diminished. Among different sizes of AuNPs, both 10 nm and 50 nm AuNPs increased spleen index, and the treatment of 50 nm AuNPs enhanced the population of MDSC. However, the expression of these cytokines was not size-dependent. Collectively, these results suggested that administration of AuNPs contributed to inflammatory responses and enhanced Th1 immunity. AuNPs induced the suppressive activity of the elevated splenic MDSC. Moreover, the biological activity of AuNPs on immune responses in vivo may not be in a size-dependent manner.