红毛五加多糖AHP-Ⅱ的荧光标记及其在小鼠腹腔巨噬细胞上的定位

Fluorescent substance labeling of Acanthopanax giraldii Hams Polysaccharide and cellular localization in mice peritoneal macrophages

目的:对红毛五加多糖AHP-Ⅱ的还原性末端进行选择性荧光标记,并观察AHP-Ⅱ与小鼠腹腔巨噬细胞结合情况。方法:利用胺化还原法对AHP-Ⅱ进行异硫氰酸荧光素(FITC)荧光标记,并通过紫外吸收光谱扫描和荧光分光光度术对偶联标记结果进行确证。通过测定多糖标记前后对小鼠腹腔巨噬细胞激活情况,检验该标记方法对AHP-Ⅱ生物学活性的影响。采用荧光显微技术和流式细胞术观察AHP-Ⅱ与小鼠腹腔巨噬细胞的结合情况。结果:AHP-Ⅱ荧光标记后的多糖得率和荧光标记效率分别为20.57%和0.809%。生物活性检测发现,AHP-Ⅱ还原性末端荧光标记后,对其激活小鼠腹腔巨噬细胞的能力没有显著影响(P>0.05)。荧光显微观察显示AHP-Ⅱ-FITC定位在小鼠腹腔巨噬细胞表面,流式细胞术检测发现在试验剂量范围内(50~200μg·m L^(-1)),巨噬细胞的荧光强度与AHP-Ⅱ-FITC加入量呈正相关,且荧光标记多糖与未标记多糖间存在竞争关系。结论:该方法可用于AHP-Ⅱ的标记研究,AHP-Ⅱ可与小鼠腹腔巨噬细胞细胞膜结合。

Objective： To selectively fluorescent labeling Acanthop anax giraldii Harms polysaccharide AHP-Ⅱ, and confirm the attachment between AHP-Ⅱ and mouse peritoneal macrophages.Methods： The amination reduction method was used in fluorescent labeling of AHP-Ⅱ.Fluorescein isothiocyanate （FITC） was linked up with the reducibility termination of AHP-Ⅱ.The result of coupling tag was confirmed by UV absorption spectra and spectrophotofluorimetry.To test the effect of the labeling method on the biological activity of AHP-Ⅱ, the ability to activate mouse peritoneal macrophage before and after polysaccharide labeling was determined.The combination status between AHP-Ⅱ and MPM was analyzed by both fluorescent microscope and flow cytometry.Results： The yield of AHP-Ⅱ-FITC and the fluorescent substitute ratio 20.57% and 0.809%, respectively.Biological activity determination showed that there was no significant change to the ability to activate MPM after AHP-Ⅱ labeled by fluorescence （P〉0.05）.The results measured with fluorescence microscope and flow cytometry showed that AHP-Ⅱ-FITC was attached on the surface of MPM and within the scope of the test dose （50-200μg·mL-1） of AHP-Ⅱ-FITC, the fluorescence intensity of MPM kept the positive correlation to the amount of AHP-Ⅱ-FITC and there was a competitive relationship between AHP-Ⅱ-FITC and AHP-Ⅱ.Conclusion： The method can be used for fluorescent labeling of the polysaccharide AHP-Ⅱ.AHP-Ⅱ can bind to MPM cytomembrane.