Effects of neutrophils alkaline phosphatase on functions of neutrophils in vitro
10.3760/cma.j.issn.0253-2727.2016.05.010
- VernacularTitle: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
- Author:
Haining LI
1
;
Wei LI
;
Jin YANG
;
Chunyan ZHANG
;
Huiyi WU
Author Information
1. 徐州医学院附属连云港医院
- Keywords:
HL-60 cells;
Alkaline phosphatase;
Cell migration assays;
Reactive oxygen species;
Apoptosis
- From:
Chinese Journal of Hematology
2016;37(5):405-411
- CountryChina
- Language:Chinese
-
Abstract:
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-related 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 CD1 lb+ 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) × 103 vs (8.00±0.78) × 103] (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,activecaspase-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.
