- 學位論文
奈米金在卵白蛋白免疫小鼠脾臟促進骨髓衍生抑制型細胞之生成
Gold Nanoparticle Enhanced the Production of Myeloid-Derived Suppressor Cells in the Spleen of Ovalbumin-sensitized Mice
摘要
奈米金在生物醫學領域的應用極具潛力，包含細胞成像、藥物傳輸、免疫治療以及光熱治療等。許多研究指出，奈米金會累積於脾臟中，暗示奈米金對免疫系統可能具有潛在的影響及毒性。文獻指出投予奈米金會影響免疫細胞的增生及其功能。然而，全身性暴露奈米金對免疫系統的潛在毒性與詳細機制的研究尚不充分，且奈米金是否影響抗原專一性免疫反應也有待釐清。因此，本論文的研究主旨在探討全身性暴露奈米金對卵白蛋白（ovalbumin；OVA）免疫小鼠之抗原專一性免疫反應及脾臟之影響。BALB/c小鼠於第1至8天，每天以靜脈注射方式投予奈米金（0.1-1 mg/kg）或磷酸鹽緩衝生理食鹽水作為對照組，於第2天給予腹腔注射OVA進行免疫，於第9天犧牲，採集各組小鼠血清以及脾臟進行後續實驗，包含血清中抗體表現量、脾臟指數（spleen index）、脾臟細胞族群、細胞激素表現量、掃描式電子顯微鏡觀察以及組織切片之免疫螢光染色觀察。實驗結果顯示，奈米金對於OVA免疫小鼠抗原專一性之抗體IgM、IgG1、IgG2a以及IgE並無影響，但造成脾臟指數顯著上升以及白髓之擴散。電子顯微鏡及X光能量散色光譜儀檢測到脾臟中有奈米金粒子。以流式細胞儀分析細胞表面抗原，發現脾臟中CD11b+Gr-1+細胞族群的比例顯著增加。將脾臟製備成單一細胞懸浮液，分別以脂多醣（lipopolysaccharide）或OVA刺激48或72小時後，以Griess反應測定一氧化氮（nitrite；NO）含量以及酵素連結免疫螢光分析法定量上清液中細胞激素之含量，結果顯示投予奈米金的組別，NO、腫瘤壞死因子（TNF）-α及干擾素（interferon；IFN）-γ表現量顯著上升。將脾臟組織切片以免疫螢光染色法，雙染檢測Gr-1和功能性之細胞激素介白素（interleukin；IL）-10、腫瘤生長因子（TGF）-β與IFN-γ，以及調節性T細胞之轉錄因子Foxp3和細胞激素IL-10，結果顯示給予奈米金之組別，位於脾臟白髓之Gr-1+IL-10+、Gr-1+TGF-β+、Gr-1+IFN-γ+以及Foxp3+IL-10+細胞數量皆顯著增加。本研究結果顯示全身性暴露奈米金，雖然對於抗原專一性之免疫反應無作用，但會引起脾臟發炎，且會誘導脾臟中具有表現細胞激素功能的骨髓衍生抑制型細胞（myeloid-derived suppressor cells）與調節性T細胞的增生。
並列摘要
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.
參考文獻
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