一种高效的FITC标记LPS的方法和其标记效率检测.docVIP

一种高效的FITC标记LPS的方法及其标记效率检测 李宇宁，曹红卫，郑江△，刘鑫，鲁永玲，王艳艳（第三军医大学附属西南医院综合实验研究中心，△为通讯作者） 【摘要】目的 探索一种的荧光素示踪内毒素（LPS）的方法 方法 将LPS解聚为单体后，在37 ℃中性溶液中用FITC标记LPS 18h，通过Ultra-4超滤离心管将小分子游离FITC及其它杂质分离纯，并用荧光分光光度计分别测定标液和纯后的FITC-LPS溶液的荧光值，将两者进行比较，测定荧光标记率。结果 本方法标记的FITC-LPS在激光共聚焦下能进行良好示踪。经荧光分光光度计检测其标记率为mol比值）。结论 FITC标记效率高，简单，为医药学实验中示踪LPS提供了一条有效的途径。 【关键词】内毒素；LPS；FITC；标记方法 R392-3 文献标识码：B An efficient method of labeling fluorescein isothiocyanate to lipopolysaccharide and the labeling efficiency detection Yuning Li,Hongwei Cao,Jiang Zheng△,Xin Liu,Yongling Lu,Yanyan Wang(Medical Research Centre,Southwest Hospital,Third Military Medical University,Chongqing 400038, China;△Corresponding Author) 【Abstract】Objective To set up a more fluorescein labeling method to trace lipopolysaccharide. Methods LPS was disaggregated into monomers and then added a surface activator, labeled with FITC in the neutral solution at 37℃ for 18h. The FITC-LPS was further purified by using a Ultra-4 millipore filter. By examination of spectrophotometer, a comparative analysis of labeling efficiency was performed between the purified solution and the raw solution. Results The end product of FITC-LPS can be detected effectively under confocal microscope, expressing fine tagging ability. This new method can reach a higher labeling rate (with a FITC to L PS ratio being 33.13:1) than the traditional method does. Conclusion The new method combining the surface activator and ultra-filter shows a higher FITC-LPS labeling efficiency, which would become into a new approach for tracing LPS in experimental research. 【Key words】Endotoxin ; Lipopolysaccharide ; Fluorescein isothiocyanate ; Labeling methodology 内毒素(endotoxin，ET)是革兰氏阴性细菌细胞壁外膜的组成成分，细菌死亡胞壁裂解后释放。内毒素主要成分为脂多糖(lipopolysaccharide，LPS)[1]。LPS与胞膜受体TLR4结合，介导炎症细胞活化，其结合分布状态是研究LPS致病机理的重要检测指标之一。越来越多的实验研究需要对LPS进行标记示踪，而商化荧光标记LPS在前期研究和众多应用回馈中效果很不理想。故本研究主在探讨一种确切有效的LPS标记方法，以解决炎症相关课题研究中示踪LPS的迫切问题。 1 材料与方法 仪器与材料 RAW264.7细胞；LPS（055: B5sigma）；FITC荧光素（sigma）；055: B5，Lot F8666，sigm