中性粒细胞碱性磷酸酶对中性粒细胞功能影响的体外研究

Effects of neutrophils alkaline phosphatase on functions of neutrophils in vitro

目的探讨中性粒细胞碱性磷酸酶（neutrophils alkaline phosphatase，NAP）对中性粒细胞迁移、活性氧（ROS）生成以及凋亡的影响。方法通过慢病毒感染人早幼粒细胞白血病细胞株HL-60获得NAP高表达细胞株，采用RT-PCR和Westernblot技术分别检测感染后细胞NAP的基因转录及蛋白表达水平。HL-60细胞经1．5％DMSO体外诱导分化为类中性粒细胞后进行相关实验：分别利用Transwell迁移实验和流式细胞术检测高表达NAP中性粒细胞的迁移及ROS生成能力；流式细胞术检测细胞凋亡；Westernblot法检测高表达NAP中性粒细胞中凋亡相关蛋白Bax、caspase-3和caspase-9的表达情况。结果经慢病毒感染的HL-60细胞通过嘌呤霉素筛选后80％以上细胞可见绿色荧光，且NAP在基因及蛋白水平表达均明显升高。HL．60细胞经1．5％DMSO体外诱导分化5d后，细胞体积变小、表面出现许多突起，核质比例降低，核仁消失，核染色质由密集趋向疏松，细胞核扭曲折叠呈杆状或分叶，CD11b＋细胞百分比明显升高。Transwell迁移实验结果显示，高表达NAP的中性粒细胞迁移细胞数多于阴性对照组[（15．30±3．65）×10^3对（8．00±0．78）×10^3，P〈0．001]。流式细胞术检测结果表明，高表达NAP的中性粒细胞胞内ROS的平均荧光强度高于阴性对照组（355．70±20．10对103．22±4．71，P〈0．001）；与阴性对照组比较，高表达NAP中性粒细胞胞凋亡率及凋亡相关蛋白Bax、active-caspase．3以及active．caspase-9的表达水平明显上调。结论NAP具有促进类中性粒细胞迁移及ROS生成、加速细胞凋亡的生物学作用。

Objective To investigate the effects ofneutrophils alkaline phosphatase （NAP） on the migration, reactive oxygen species （ROS） generation and apoptosis of neutrophil-like differentiated HL-60 cells. Methods NAP was overexpressed in HL-60 cells via transfecting coding sequence of NAP by lentivirus. The effectivity of NAP overexpression was confirmed by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting. HL-60 cells were differentiated into neutrophil-like cells by exposure to 1.5% DMSO. The migration and ROS generation of neutrophil- like cells with NAP overexpression were detected by transwell migration test and flow cytometry, respectively. Cell apoptosis were detected by flow cytometry. The expression of apoptosis-rclated protein Bax, caspase-3 and caspase-9 in neutrophil-like cells were observed by Western blotting after NAP overexpression. Results Over 80% HL-60 cells presented green fluorescence after GFP-NAP infection by lentivirus and screening by puromycin. In addition, the levels of both gene and protein of NAP were up-regulated in these cells. After 5- day culture with 1.5% DMSO, the bulk of induced cell was smaller and the surface appeared many bumps and irregular shape. The ratio of nuclear and cytoplasmic decreased and nucleolus disappeared. The nuclear chromatin changed from dense to loose and the nuclear morphology appeared to be rod and segmented. The percentage of CD 11 b＋ cell increased from 26.25% to 98.55%. The transwell migration test showed that the number of migrated cells was higher in neutrophil-like cells with NAP overexpression compared with the negative control [ （ 15.30±3.65 ） ×10^3 vs （ 8.00±0.78 ） × 10^3 ] （P〈0.001 ）. Results of flow cytometry suggested that the mean fluorescence intensity （MFI） of intracellular ROS was significantly higher in neutrophil-like cells with NAP overexpression compared with the negative control （355.70±20.10 vs 103.22±4.71） （P〈 0.001 ）. In addition, Western blotting showed that the expressions of apoptosis-related protein Bax, active- caspase-3 and active-caspase-9 were all up-regulated in neutrophil-like cells with NAP overexpression compared with the negative control. Conclusion NAP could promote the migration and ROS generation of neutrophil-like cells and accelerate the cell apoptosis.