用基因标记技术实时观察中性粒细胞吞噬病原体

Phagocytosis mechanism of neutrophilic granulocyte with green fluorescent protein gene in living cell

目的 试图建立一种快速、直接判断中性粒细胞的吞噬、杀菌功能的标准方法 ,并对该方法进行分析与评价。方法 将带有绿色荧光蛋白 (GFP)基因的PRSETB质粒转化为大肠埃希菌BL2 1 ,利用BL2 1 高效表达GFP后发出强烈绿色荧光的特点 ,用电荷耦合器件 (CCD)耦合荧光显微镜连续记录中性粒细胞对大肠埃希菌粘附、内吞、消化和残体外排的全过程。结果 粘附过程 :BL2 1 在中性粒细胞膜表面停顿约 1 5s(1 5 0± 0 15s) ,为抗原识别过程 ;内吞过程 :中性粒细胞膜内陷、伸出伪足将大肠埃希菌包裹 ,并向细胞中心移动 ,持续时间可达 132s(132± 14s) ;消化过程 :当大肠埃希菌进入中性粒细胞胞体后 ,其杆状体逐渐变短 ,GFP发出的荧光强度逐渐变弱 ,持续时间约为 35 0s(35 0± 32s) ;残体外排过程 :可观察到溶酶体残体由中性粒细胞中心向胞膜运动 ,最后排出 ,持续时间约为 140s(140± 2 7s)。结论 该方法利用光子学显像和基因标记相结合的技术可以实时、连续观察中性粒细胞对大肠埃希菌的吞噬杀菌过程 ,对临床治疗有重要意义。

Objective To develop a novel method to trace the phagocytosis process of neutrophilic granulocyte in real time with a photonic technique combined with gene marker.Methods Escherichia coli BL 21 was transformed with plasmid P RSETB that carried green fluorescent protein (GFP) gene. The transformed BL 21 efficiently expressed GFP and emitted a green fluorescence as exciting with 390 nm blue light. The whole process of neutrophilic granulocyte phagocytosis was recorded with charge coupled device (CCD) coupled fluorescent microscopy. Results Adhesion phase spent 1.5 seconds (1.50±0.15 s). Endocytosis phase spent 132 seconds (132±14 s). Digestion phase spent 350 seconds (350±32 s). Exocytosis phase took 140 seconds (140±27 s). The configuration properties of four phases of neutrophilic granulocyte phagocytosis were recorded. Conclusion The combined technique of photonics and gene marker could trace the phagocytosis process of neutrophilic granulocyte in real time.