体外诱导糖尿病小鼠模型中性粒细胞胞外诱捕网的研究

Study on the Induction of Neutrophil Extracellular Traps from Diabetic Mice in vitro

目的建立佛波酯(phorbol 12-myristate 13-acetate,PMA)诱导糖尿病小鼠骨髓来源中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)形成的模型,为研究糖尿病足溃疡NETs产生和调节机制奠定基础。方法提取链脲佐菌素(streptozocin,STZ)诱导的糖尿病小鼠骨髓来源中性粒细胞,使用100、200、400nmol/L的PMA刺激细胞3~7h。采用核酸染料Sytox Green染色胞外DNA,通过激光共聚焦显微镜检测各组网状结构的形成、荧光免疫组化法检测瓜氨酸化组蛋白H_(3)(citrulline histone H_(3),Cit-H_(3))的表达、荧光探针测定细胞内活性氧(reactive oxygen species,ROS)水平,电子扫描显微镜下观察NETs形成情况。结果各组PMA刺激中性粒细胞,刺激5h形成少量NETs,6h时形成大量网状NETs结构,其中100nmol/L PMA处理组有明显NETs生成,7h时各组NETs开始消退;同时100nmol/L PMA刺激中性粒细胞6h时Cit-H_(3)的表达以及ROS水平显著升高;扫描电子显微镜可见NETs网状结构。结论100nmol/L PMA刺激STZ诱导的糖尿病小鼠骨髓中性粒细胞6h,可显著促进Cit-H_(3)表达和ROS生成,诱导NETs生成,可作为研究糖尿病小鼠NETs研究的模型。

Objective To establish a bone marrow-derived neutrophil extracellular traps(NETs)model induced by phorbol 12-myristate 13-acetate(PMA)in diabetic mice and to lay the foundation for studying the NETs formation and regulation mechanism in dia-betic foot ulcers.Methods Bone marrow-derived neutrophils were isolated from streptozocin(STZ)-induced diabetic mice and stimu-lated with PMA(100,200,400nmol/L)for 3-7h.The formation of the network structure in each group was detected by a laser confocal microscope after staining with the Sytox Green.The expression of citrullinated histone H_(3)(Cit-H_(3))was checked by fluorescence immu-nohistochemistry.The intracellular reactive oxygen species(ROS)levels were determined by fluorescent probes.NETs formation was ob-served by the scanning electron microscope.Results NETs was formed when Bone marrow-derived neutrophils were induced with PMA for 5h and NETs formation was obviously increased for 6h and the NETs began to fade for 7h.Especially,the 100nmol/L PMA treatment group could significantly promote the formation of NETs.And the expression of Cit-H_(3) and the level of ROS increased significantly in neutrophils induced with 100nmol/L PMA for 6h.What′s more,the NETs network structure was visible by the scanning electron micro-scope.Conclusion 100nmol/L PMA stimulation of bone marrow-derived neutrophils from STZ-induced diabetic mice for 6h could significantly promote Cit-H_(3) express and ROS production and induce NETs formation,and it could be as a NETs research model for dia-betic mouse.