摘要
为确定树舌多糖在小鼠脾淋巴细胞上的定位,应用胺化还原法在树舌多糖(GAP)的还原性末端连接异硫氰酸荧光素(FITC),用荧光分光光度计测定其荧光取代度,xCELLigence RTCA DP全自动实时细胞分析仪检测GAP荧光标记前后生物学稳定性。流式细胞仪检测小鼠脾T淋巴细胞、B淋巴细胞和NK细胞中的GAP荧光强度,荧光显微镜观察GAP在小鼠脾淋巴细胞中的定位。结果显示:在荧光标记GAP(FITC-GAP)前后的细胞毒活性不变,并且其荧光取代率为0.90%;流式细胞仪检测发现小鼠脾T淋巴细胞、B淋巴细胞和NK细胞上的荧光信号强度与FITC-GAP加入量成正相关,荧光显微照片显示GAP定位于小鼠脾淋巴细胞表面并可转运至细胞核。
In order to confirm cellular localization on splenic lymphocytes of the mice, fluorophores was used to polysaccharide from Ganoderma applanatum （GAP）and fluorescence signal was detected indirectly to trace GAP. The reductive amination method was used in fluorescently tagged of Ganoderma applanatum polysaccharide （GAP）. Fluorescein isothiocyanate was linked with reducibility terminatio of GAP,and fluorescent substitute ratio is detected by fluorescence spectrophotometer. The biological stability of GAP before and after fluorescently-tagged was measured by xCELLigence RTCA DP automatic real-time cell analyzer. The fluorescence signal of T lymphocytes, B lymphocytes and natural killer cell of spleen were detected by flow cytometry, and cellular localization was observed by fluorescence microscope. The results showed that there is no significant changes for the cytotoxic activity in vitro before and after fluorescent labeling of GAP,and its fluorescent substitute ratio is 0.90%. The results flowing cytometry showed that fluorescence signal could be detected by each FITC-GAP experimental group of T lymphocytes, B lymphocytes and natural killer cell of mice, and fluorescence signal intensity increased with the increased FITC-GAP concentration. Observed by fluorescence microscopy, FITC-GAP both surface and intracellular. GAP was located on the cell surface and transported to the cell nucleus of splenic lymphocytes for mice.
出处
《中国农业大学学报》
CAS
CSCD
北大核心
2013年第1期147-152,共6页
Journal of China Agricultural University
基金
吉林省世行贷款农产品质量安全项目(2011-Y18)
关键词
树舌
多糖
荧光标记
脾细胞
细胞定位
Ganoderma applanatum
polysaccharide
fluorescent labeling
spleen lymphocytes
cellular localization
