基于敏感细胞识别的食源性致病菌毒力分析模型研究

Sensitive epithelial cells HT-29, which could specific recognize pathogens lipopolysaccharide LPS and stable express enhanced green fluorescent protein,were built. TLR4 expression and the fluorescence signal conversion, as well as proinflammatory Factor Expression Induced by different foodborne pathogens were researched by using this sensitive cell model. Both the conductive elements with the extracellular matrix of the covalent binding and graphene/gold nanoparticle composite synergistic effect made cell effectively fixed and fast electron transfer between the electrode substrate. Experiments were conducted with the combination of theoretical calculation to study the behavior regularity of the intrinsic relationship between cell electrochemical nature and molecular configuration, conductive, ionic liquid, and physicochemical properties of grapheme/gold nanomolecular conductance, and explore the effect nature of molecular structures on the regularity of the influence. Based on nanocomposite material/conductive polymer/ionic liquid/extracellular matrix preparation Colorectal cancer cell-based biosensor evaluation model was developed by using Salmonella as an example. Research was deeply carried out to clarify the behavior of the cell, conductive elements and structure properties of ionic liquids, and electrochemical properties relations, and put forward cell electrochemical behavior infected by pathogen, and signal transduction mechanism of composite materials working on cell response. The research results were applied into the process of food processing and quality control, which would realize the foodborne pathogen rapid evaluation and timely warning, and make our country come into the international forefront in food safety evaluation research areas, and promote international academic exchanges and personnel training.

构建能特异性识别致病菌脂多糖的敏感肠道上皮细胞HT-29，研究不同食源性致病菌对敏感细胞荧光信号转化、TLR4表达以及其对细胞促炎因子诱导的影响规律，研究亲生物性离子液体和细胞外基质胶原纤维混合凝胶的形成机制，构建细胞三维生长最佳微环境，结合石墨烯金纳米聚合物形成的功能性复合膜，建立具有精细电子传导功能的细胞传感检测体系。通过对模型细胞行为、基底材料的结构性质和电化学传感分析性能三者关系的深入研究，提出细胞受致病菌侵染后的电化学行为变化及其规律性，结合细胞内荧光强度变化和细胞炎症因子的诱导和表达，建立系统分析食源性致病菌及其毒力的细胞传感评价模型，为完善食品安全预警和控制体系提供基础数据和理论指导，使我国在食品安全控制与预警方面的研究水平进入国际前沿。

以食源性致病菌及其毒力因子为研究对象，基于食源性致病菌脂多糖LPS毒性通路，将电化学检测、重组蛋白质粒的构建、荧光报告基因转染以及磁性分子印迹等手段相结合构建一系列新型灵敏的食源性致病菌及毒力筛查的传感新方法与新原理。构建基于纳米复合材料/导电分子/细胞外基质的细胞传感评价模型，应用于鼠伤寒沙门氏菌 ATCC 14028、大肠杆菌 FSCC 149002、痢疾志贺氏菌 ATCC3313等检测，形成细胞相容性分子设计路线，获得14种致病菌毒力对细胞荧光及电化学反应的剂量效应关系，所构建的纳米复合材料介导的电化学细胞传感器、实现了超灵敏检测食源性致病菌脂多糖LPS引起的电化学阻抗变化，为探究LPS引起的细胞电化学行为和细胞行为机制提供了基础；筛选致病菌信号分子和毒力基因，合成纳米颗粒增强检测信号，实现了基于核酸探针识别食源性致病菌以及磁性材料对食源性致病菌的高效富集；基于脂多糖LPS的毒性作用通路，构建特异性识别食源性致病菌脂多糖LPS、稳定高表达荧光转染细胞模型，结合胞内荧光检测，实现脂多糖LPS细胞评价的可视化，克服了传统检测手段只能对脂多糖LPS进行定量而无法反映其毒力大小的缺点，实现了直接评价致病菌毒力因子脂多糖LPS毒力大小的目标，突破了传统检测的被动性，克服已知菌的检测中无法识别和判断未知菌株及菌株毒力大小的技术瓶颈，为实现食源性致病菌非靶向筛查奠定理论基础，也为食源性致病菌毒力因子脂多糖LPS毒力大小的判定及食品安全性筛查提供一种崭新的方法；在机制上进一步解释不同致病菌LPS诱导差异性，基于细胞代谢组学研究脂多糖LPS对细胞代谢影响，探究食源性致病菌脂多糖LPS对肠道的毒性作用机制及其介导疾病的作用机制，通过对细胞溶出物的质谱组学分析，确证脂多糖LPS对人结肠癌细胞HT-29和肥大细胞RBL2H3的胞内代谢影响规律与机制，为进一步阐释脂多糖LPS毒性作用机理提供了依据，为阐明LPS介导的疾病的潜在机制提供了启发性指导，为实现食源性致病菌对肠道毒性作用的早期预警提供了可能。