第一部分 纳米SiO₂颗粒协同IgE介导的肥大细胞活化加重过敏性炎症的研究

背景：过敏性疾病已成为全球性的健康问题。大气颗粒物、柴油废气颗粒和环境烟草烟雾等空气污染物的增加都大大加重了环境暴露带来的健康负担，空气污染物已成为促进过敏性疾病发生发展的重要环境因素。大气颗粒物的来源和组成多样，包含大量生物和非生物活性成分，两者相互作用使得大气颗粒物诱发过敏性疾病的机制十分复杂。目前，关于大气颗粒物中不同粒径的非生物活性成分对过敏性疾病影响的研究有限，其生物学效应和分子机制尚未完全阐明。

目的：探讨不同粒径大小的非生物活性颗粒物与过敏性疾病之间的效应关系和可能的分子机制，科学评价大气颗粒物中非生物活性成分对过敏性疾病的影响。

方法：本研究选取Al₂O₃，TiO₂，Fe₂O₃和SiO₂四种包含纳米（nm）和微米（μm）两个粒径大小的大气颗粒物中常见矿物成分作为代表，通过培养小鼠骨髓来源肥大细胞，建立卵清蛋白诱导的过敏性哮喘小鼠颗粒物暴露模型和花粉致敏小鼠颗粒物暴露模型，以评估颗粒物暴露的生物学效应以及可能的分子作用机制。

结果：

不同粒径大小颗粒物的高浓度暴露均具有明显的细胞毒性。20 nm SiO₂颗粒暴露能够协同增强IgE介导的致敏肥大细胞的活化效应，并具有一定的浓度效应关系。

低剂量纳米SiO₂颗粒暴露虽未引起正常小鼠肺部大量炎性细胞浸润、气道结构明显破坏和明显纤维化改变，但仍能增加大气道黏液分泌和提高血清组胺水平。

纳米SiO₂颗粒暴露能提高OVA致敏哮喘小鼠细胞因子IL-4和IL-6的分泌，募集肥大细胞到肺组织炎症部位，增强肥大细胞活化和组胺分泌，增加气道黏液分泌，增强气道高反应性，加重过敏性气道炎症反应。

纳米SiO₂颗粒暴露进一步破坏花粉致敏小鼠鼻黏膜上皮的完整性，增加杯状细胞和黏液分泌，提高血清IL-4和IL-6的水平，进一步加剧Th1/Th2细胞失衡，并导致机体以烟酸和烟酰胺等为代表的代谢机制紊乱，加重过敏性炎症反应。

肥大细胞可以摄取纳米SiO₂颗粒，但并未影响肥大细胞FcεRI的表达和结合IgE的能力，而是通过增加ERK1/2的磷酸化水平，激活MAPK信号通路协同增强IgE介导的致敏肥大细胞的活化效应。

结论：研究结果表明纳米SiO₂颗粒暴露可能通过激活MAPK信号通路协同增强IgE介导的致敏肥大细胞活化效应，增加组胺、IL-4和IL-6等炎性因子分泌，加重过敏性气道炎症反应。本研究结果为大气颗粒物诱发过敏性疾病的防治策略提供了理论依据。

第二部分 二球悬铃木花粉过敏原蛋白组分profilin的鉴定、表达及致敏性研究

背景：悬铃木属花粉是我国春季最常见的过敏原之一，已成为诱发过敏性呼吸道疾病的重要因素。但有关二球悬铃木花粉致敏蛋白组分的研究十分有限，还存在较多的过敏原蛋白组分尚未被鉴定出来。悬铃木属花粉过敏与植物性食物过敏之间存在很强的相关性，profilin可能是二球悬铃木花粉中一种重要的过敏原致敏蛋白组分。然而，致敏蛋白组分profilin的分子特征和致敏性尚未得到充分阐述。

目的：对二球悬铃木花粉致敏蛋白组分profilin进行鉴定和表征。

方法：本研究将致敏蛋白组分profilin的编码序列通过扩增、克隆，构建重组质粒，然后在大肠杆菌BL21细胞中表达，通过镍亲和层析分离纯化。对致敏蛋白组分profilin进行氨基酸序列比对、分子理化性质和蛋白质结构模拟分析。利用二球悬铃木花粉过敏患者血清通过酶联免疫吸附试验（ELISA）、蛋白免疫印迹实验（WB）和嗜碱性粒细胞活化试验（BAT）等对重组致敏蛋白组分profilin的致敏性和交叉反应性进行评估。

结果：

重组致敏蛋白组分profilin含有396个核苷酸，可编码131个氨基酸，分子量约为14 kDa。其氨基酸序列与其他吸入性过敏原和食物过敏原中的profilin具有较高的同源性，二级结构包含3个α-螺旋和7个β-折叠。分子理化性质分析表明其是一种酸性、稳定、相对耐热和相对保守的蛋白质。

利用二球悬铃木花粉过敏患者血清通过ELISA、WB和BAT等实验分析了重组致敏蛋白组分profilin的IgE结合活性，证实其具有相对较高的致敏率（76.9%, 30/39），且与黑杨花粉过敏原蛋白组分Pop n 2存在显著的交叉反应。

结论：本研究在分子和免疫水平上对致敏蛋白组分profilin进行了鉴定和表征，证实其是二球悬铃木花粉中的主要致敏蛋白组分之一。研究结果为二球悬铃木花粉的过敏原组分解析诊断和个体化特异性免疫治疗疫苗的研制提供了理论依据。

Part I. Nano-silica particles synergistically IgE-mediated mast cell activation exacerbating allergic inflammation

Background: Allergic diseases have become a global health problem. The worsened air quality accompanied by increased air pollutants such as atmospheric particulate matter, diesel exhaust particles, and environmental tobacco smoke has dramatically increased the health burden brought by environmental exposure. Air pollutants have become an important environmental factor in developing allergic diseases. The sources and compositions of atmospheric particulate matter are diverse, including many biologically and non-biologically active components. The interaction between the components complicates the mechanism of atmospheric particulate matter-induced allergic diseases. However, studies are limited on the effects of inorganic components and particulate matter with different particle sizes in atmospheric particulates on allergic diseases, and the possible molecular mechanism of inducing allergies has not been thoroughly studied.

Objective: To explore the relationship and possible molecular mechanism between the non-biologically active components in different particle sizes and allergic effects, and evaluate the effects of non-biologically active components in the atmospheric particulate matter on allergic disease scientifically.

Methods: This study selected four standard mineral components in atmospheric particulate matter containing nanometer (nm) and micrometer (μm) particle sizes as representatives, including Al₂O₃, TiO₂, Fe₂O₃, and SiO₂. By culturing mouse bone marrow-derived mast cells, establishing an ovalbumin-induced allergic asthma mouse particulate matter exposure model, and pollen-sensitized mouse particulate matter exposure model to evaluate the biological effects of particulate matter exposure and possible molecular mechanisms.

Results：

(1) Exposure to high concentrations of particles with different particle sizes had obvious cytotoxicity. Exposure to 20 nm SiO₂ particles could synergistically enhance the activation effect of IgE-mediated mast cells with a particular concentration-effect relationship.

(2) Although the exposure of low-dose nano-SiO₂ particles did not cause significant damage to the pulmonary airway structure, infiltration of many inflammatory cells, and noticeable fibrotic changes in normal mice, it still increased mucus secretion in the large airways and serum histamine levels.

(3) Nano-SiO₂ particles exposure increased the secretion of cytokines IL-4 and IL-6 in OVA-induced asthmatic mice, recruited mast cells to inflammatory sites in lung tissue, enhanced mast cells activation and histamine secretion, increased airway mucus secretion, enhanced airway hyperresponsiveness, and aggravated allergic airway inflammation.

(4) Nano-SiO₂ particles exposure aggravated the damage of nasal mucosa epithelial cells in pollen-sensitized mice, increased goblet cells and mucus secretion, increased serum IL-4 and IL-6 levels, and further aggravated the imbalance between Th1/Th2 cells, led to the disorder of the metabolic mechanism represented by niacin and nicotinamide and aggravated allergic inflammatory response.

(5) Mast cells could uptake nano-SiO₂ particles, which did not affect the expression of FcεRI and the ability to bind IgE in mast cells but increased the phosphorylation level of ERK1/2 and activated the MAPK signaling pathway to enhance the activation effect of IgE-mediated mast cells synergistically.

Conclusion: The results indicated that the exposure of nano-SiO₂ may synergistically enhance the activation effect of IgE-mediated mast cells by activating the MAPK signaling pathway, increasing the secretion of inflammatory factors such as histamine, IL-4, and IL-6, and aggravating allergic airway inflammation. Our research provided a theoretical basis for preventing and treating allergic diseases induced by atmospheric particulate matter.

Part II. Identification, expression, and sensitization study of the profilin from Platanus acerifolia pollen

Background: Platanus acerifolia (P. acerifolia) is one of the most common allergens in spring in China, and it has become an important factor inducing allergic respiratory diseases. However, studies on the allergenic components of P. acerifolia pollen are very limited, and many unknown components have not been identified. There is a strong correlation between P. acerifolia pollen and plant food allergy, suggesting profilin may be an important allergenic component in P. acerifolia pollen. However, the molecular characterization and sensitization of the profilin have not been fully studied yet.

Objective: The study aimed to identify and characterize the allergenic component profilin from the P. acerifolia pollen.

Methods: The coding sequence of profilin was amplified, cloned, and then expressed in Escherichia coli BL21 cells and purified by nickel affinity chromatography. The amino acid sequence alignment, molecular physicochemical properties, and protein structure simulation analysis were performed. The allergenicity and cross-reactivity were assessed by enzyme-linked immunosorbent assay (ELISA), western blot (WB), and basophil activation test (BAT) using the sera from P. acerifolia allergic patients.

Result:

(1) The cDNA sequence of the allergenic component profilin was cloned with a 396 bp open reading frame coding for 131 amino acids, with a molecular weight of about 14 kDa. Its amino acid sequence had a high homology identity with profilin in other aeroallergens and food allergens, and the predicted structure consisted of 3 α-helixes and 7 β-sheets. Physicochemical analysis indicated that the profilin was an acidic, stable, relatively thermostable, and relatively conserved protein.

(2) The IgE-binding activity of the recombinant profilin was determined by ELISA, WB, and BAT using the serum of P. acerifolia pollen allergic patients, which suggested a relatively high IgE sensitization rate (76.9%, 30/39). And observed a significant cross-reactivity with Pop n 2, the profilin from Populus nigra, in the IgE-inhibition assay.

Conclusion: In this study, the allergenic component profilin was identified and characterized at the molecular and immunological levels and confirmed as one of the major component allergens in P. acerifolia pollen. The results provided useful information for component-resolved diagnostics and the development of individualized specific immunotherapy vaccines for P. acerifolia pollen allergic patients.