论文部分内容阅读
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 fluorescently label the reducing end of AHP-Ⅱ and observe the binding of AHP-Ⅱ to mouse peritoneal macrophages. Methods: AHP-Ⅱ was labeled with fluorescein isothiocyanate (FITC) by amination-reduction method, and the results of coupling labeling were confirmed by ultraviolet absorption spectroscopy and fluorescence spectrophotometry. By measuring the activation of mouse peritoneal macrophages before and after polysaccharide labeling, the effect of this labeling method on the biological activity of AHP-Ⅱ was tested. The binding of AHP-Ⅱ to mouse peritoneal macrophages was observed by fluorescence microscopy and flow cytometry. Results: The yield of polysaccharide and fluorescent labeling efficiency after AHP-Ⅱ fluorescence labeling were 20.57% and 0.809% respectively. The results of bioassay showed that AHP-II reduced the ability of activating mouse peritoneal macrophages (P> 0.05). Fluorescence microscopy showed that AHP-Ⅱ-FITC was located on the surface of peritoneal macrophages in mice. Flow cytometry showed that fluorescence of macrophages was detected in the range of 50 ~ 200 μg · mL -1, There was a positive correlation between the intensity and the amount of AHP-Ⅱ-FITC addition, and there was competition between fluorescently labeled polysaccharides and unlabeled polysaccharides. Conclusion: This method can be used for labeling of AHP-Ⅱ, and AHP-Ⅱ can bind to the membrane of mouse peritoneal macrophages.