目的 通过定性和定量分析蓝氏贾第鞭毛虫（简称贾第虫）滋养体对中性粒细胞胞外诱捕网的诱导能力。方法 采用中性粒细胞分离试剂盒提取健康人血中的中性粒细胞,与等数量贾第虫滋养体（均为4 × 10⁵）加入2 ml M199培养基中分别培养30、60、120、180、240 min,收集各时间段的培养上清,使用PicoGreen染色法定量检测上清双链DNA（dsDNA）的浓度,ELISA检测上清中髓过氧化物酶（MPO）的含量;另取中性粒细胞与贾第虫滋养体（均为4 × 10⁵/ml）各200 μl为实验组,加至24孔板中,37 ℃混合培养4 h后,用蓝色荧光标记DNA、绿色荧光标记瓜氨酸化组蛋白H3、红色荧光标记MPO,激光共聚焦显微镜下观察中性粒细胞胞外诱捕网的形成情况;阳性对照组为100 nmol/L佛波酯处理的中性粒细胞,阴性对照组为未处理的中性粒细胞。应用SPSS 19.0软件进行统计学分析,数据用独立样本t检验进行比较。结果 分离获得的健康人血中性粒细胞纯度达75%以上,镜下可观察到核呈杆状或分叶状的中性粒细胞。PicoGreen染色法检测结果显示,中性粒细胞和贾第虫滋养体等数量混合30、60、120、180、240 min后,上清中dsDNA吸光度（A₅₃₀值）分别为7.30 ± 1.12、11.15 ± 1.10、21.69 ± 2.71、26.35 ± 2.26、29.29 ± 3.27,均高于阴性对照组的3.79 ± 0.48,且dsDNA浓度随着培养时间的延长而升高（P < 0.05）。ELISA检测结果显示,阴性对照组、实验组和阳性对照组上清中MPO含量的A₅₃₀值分别为0.209 4 ± 0.018 2、0.611 5 ± 0.060 7、0.721 7 ± 0.087 9,实验组的MPO含量高于阴性对照组（P < 0.05）。激光共聚焦显微镜下观察到实验组的中性粒细胞和贾第虫滋养体之间有红色和绿色网状荧光物质存在,其中含有大量组蛋白和MPO。结论 贾第虫滋养体在体外具有刺激中性粒细胞释放胞外诱捕网的能力。

Objective To study the induction of neutrophil extracellular traps (NETs) by Giardia lamblia using qualitative and quantitative methods. Methods Neutrophils were collected from blood samples of a healthy participant by using isolation reagent kit, and cultured with G. lamblia (4 × 10⁵/ml) in M199 medium for 30, 60, 120, 180 or 240 min. The concentration of double-stranded DNA (dsDNA) in the supernatant at the defined time points was determined by PicoGreen staining method. The concentration of myeloperoxidase (MPO) in the supernatant was assayed by ELISA. In the test group designed, 200 μl of neutrophils was added with 200 μl of G. lamblia (both 4 × 10⁵/ml) into 24-well plates and cultured at 37 ℃ for 4 h. Neutrophils producing extracellular trap nets were double-stained with anti-H3Cit and anti-MPO fluorescence. For visualization, DNA was labeled with blue fluorescence, citrullinated histone H3 protein was labeled with green fluorescence, and MPO was labeled with red fluorescence. Laser confocal microscopy was used to observe the formation of NETs. The positive group was set by using the neutrophils treated with 100 nmol/L phorbol ester, while the negative control group was the untreated neutrophils. SPSS software was used for statistical analysis, and the experimental data were analyzed with independent sample t-test. Results The purity of neutrophils isolated from the blood was over 75%. The neutrophils with rod-shaped or lobulated nuclei could be observed under a microscope. The fluorescence intensities after mixing the neutrophils with Giardia (4 × 10⁵/ml) for 30, 60, 120, 180, and 240 min were 7.30 ± 1.12, 11.15 ± 1.10, 21.69 ± 2.71, 26.35 ± 2.26, and 29.29 ± 3.27, respectively, while those in the negative control group and the positive control group were 3.79 ± 0.48 and 35.78 ± 2.83, respectively. The level of dsDNA in the supernatant of the test group was higher than that of the negative control group, and the content of dsDNA increased with prolonged culture time(P < 0.05). ELISA results showed that the absorbance related to the MPO content in the supernatant of the negative control group, test group and positive control group were 0.209 4 ± 0.018 2, 0.611 5 ± 0.060 7, and 0.721 7 ± 0.087 9, respectively. The MPO content of the test group was higher than that of the negative control group (P < 0.05). Laser confocal microscopy revealed presence of a reticular substance between neutrophils and Giardia, which was enriched with histone and MPO. Conclusion Giardia trophozoites have the potential to activate neutrophils releasing extracellular traps in vitro.