| Summary: | 碩士 === 國立臺灣大學 === 獸醫學研究所 === 106 === Gold nanoparticles (AuNP) are promising nanomaterials used for various biomedical purposes such as cell imaging, drug delivery, immunotherapy, photothermal therapy, etc. Previous studies reported that AuNP were accumulated in the spleen in vivo, raising concerns on potential immunotoxicity. Although accumulating evidence demonstrates that exposure to AuNP affects the proliferation and functions of immune cells in a number of in vitro and in vivo models, the detailed immunomodulatory and/or immunotoxic effects of AuNP remain to be more comprehensively characterized. In addition, little is known pertaining to the effects of AuNP on the antigen-specific immune response. Hence, the objective of this thesis study is to investigate the effect of AuNP on antigen-specific immune response and the function of spleen in ovalbumin (OVA)-sensitized BALB/c mice. Mice were either left untreated (naïve; NA), or daily administered with AuNP (0.1-1 mg/kg) and/or vehicle (phosphate-buffered saline) by intravenous administration from day 1 to day 8. Except for the NA group, the mice were sensitized with OVA by intraperitoneal injection on day 2. All mice were sacrificed on day 9. Serum samples were collected and their spleens were harvested for further experiments. The level of OVA-specific antibodies in the serum was quantified by ELISA. The spleen index was calculated as the spleen weight divided by body weight. Single-cell splenocyte cultures were prepared and stimulated with lipopolysaccharide (LPS) and OVA for 48 and 72 h, respectively. The production of cytokines in the supernatant was quantified by ELISA. The secretion of nitrite oxide (NO) was measured by using Griess reagent. The cellularity of splenocytes was examined by flow cytometry. The number of splenic Foxp3+IL-10+, Gr-1+IFN-γ+, Gr-1+IL-10+ and Gr-1+TGF-β+ cells were detected by immunofluorescent staining. Scanning electron microscopy and energy dispersive X-ray spectroscopy were conducted for the detection of AuNP in the spleen section of AuNP-treated mice. The results showed that AuNP treatment did not affect the serum production of OVA-specific IgE, IgM, IgG1 and IgG2a. However, AuNP treatment significantly increased the spleen index and the population of CD11b+ cells in OVA-sensitized mice, whereas the number of CD4+, CD8+ and B220+ cells was unaffected. In addition, the diffusion of white pulps in AuNP-treated spleen was observed in H&E staining. Results from scanning electron microscopy and energy dispersive X-ray spectroscopy detected the presence of AuNP in the spleen. AuNP treatment markedly enhanced the production of NO and tumor necrosis factor (TNF)-α by LPS-stimulated splenocytes. The expression of interferon (IFN)-γ by splenocytes stimulated with OVA was also enhanced in AuNP-treated mice. Flow cytometric analysis of the splenic cellularity revealed an increased population of CD11b+Gr-1+ cells. The number of Foxp3+IL-10+, Gr-1+IFN-γ+, Gr-1+IL-10+ and Gr-1+TGF-β+ cells in the white pulps of spleens was significantly increased in AuNP-treated groups. Collectively, these results demonstrated that systemic exposure to AuNP did not affect antigen-specific antibody production, but caused an inflammatory status in the spleen, as evidenced by the increased spleen index and expression of NO and pro-inflammatory cytokines. In addition, the elevated population of splenic CD11b+Gr-1+, Foxp3+IL-10+, Gr-1+IFN-γ+, Gr-1+IL-10+ and Gr-1+TGF-β+ cells indicated that AuNP enhanced the development of functional myeloid-derived suppressor cells and regulatory T cells.
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