荧光分子扩散动态测量在肥大细胞脱颗粒体外检测中的应用

 目的：建立基于荧光分子扩散动态测量的肥大细胞脱颗粒的体外快速评估方法。方法：分别采用β-己糖苷酶法、扫描电镜法及光栅图像关联光谱法（raster image correlation spectroscopy, RICS）对稳定表达CD63-绿色荧光蛋白（green fluorescent protein，GFP）的大鼠肥大细胞株RBL-2H3的脱颗粒过程进行评价，比较不同检测方法的灵敏度。结果：β-己糖苷酶法的检测灵敏度为5 mg/L，扫描电镜法的检测灵敏度均为3.9×10-2 mg/L，RICS方法也在3.9×10-2 mg/L即可检测到扩散系数的降低，并与未给药组有显著差异。结论：RICS方法比传统β-己糖苷酶法更灵敏，虽然与电镜方法灵敏度相当，但该方法较扫描电镜法简便易行成本低，易操作，且所观察的指标为扫描电镜所不能做到的活体动态观测，能在细胞脱颗粒早期即可判定过敏反应的发生，具有传统方法不具备的优势，有潜力成为制药企业质量控制的标准方法，或临床过敏性物质筛查的有利工具。

Detection of mast cell degranulation by fluorescent molecular diffusion dynamic measurement

AIM：To establish a rapid in vitro method for mast cell degranulation tracing by raster image correlation spectroscopy (RICS). METHODS：RBL-2H3, a basophilic granulocyte mast cell line transfected with CD63-GFP plasmid, was used for evaluating the methods, including β-hexosaminidase (HEX) colorimetric assay, scanning electron microscopy (SEM) and RICS in the detection of mast cell degranulation. The sensibilities of these methods were compared. RESULTS：The sensitivities of β-HEX colorimetric assay and SEM were 5 mg/L and 3.9×10-2 mg/L, respectively. RICS detection showed obvious decrease in the diffusion coefficient at dose of 3.9×10-2 mg/L. CONCLUSION：Fluorescent molecular diffusion dynamic measurement can be used for rapid tracing of allergic substances in vitro. According to the results, RICS can achieve nearly the same extent of sensitivity as the SEM does and is far more sensitive than β-HEX colorimetric assay. Compared with SEM, RICS has several advantages: it is faster, simpler and cheaper; it can be used in living cells; it is more suitable for rapid in vitro allergenic compounds tracing. Therefore, RICS is applicable in clinic allergic antigen screening and may also be used in pharmaceutical quality control.