尿激酶型纤溶酶原激活物受体基因对中性粒细胞活化及凋亡的影响  

Effect of plasminogen activator urokinase receptor gene on the activation and apoptosis of neutrophil

作　　者：陈娟[1] 张瑞仙 谢敏 丁湫瑾 李菁[3] Chen Juan;Zhang Ruixian;Xie Min;Ding Qiujin;Li Jing(Department of Rheumatology and Clinical Immunology,the Second Affliated Hospital of Kunming Medical University,Kunming 650101,China;Department of Disease Control and Prevention,the First People′s Hospital of Yunnan Province,Kunming 650101,China;Department of Rheumatology and Clinical Immunology,Peking Union Medical College Hospital,Beijing 100730,China)

机构地区：[1]昆明医科大学第二附属医院风湿免疫科,昆明650101 [2]云南省第一人民医院疾病预防控制科,昆明650101 [3]北京协和医院风湿免疫科,北京100730

出　　处：《中华医学杂志》2024年第11期877-882,共6页National Medical Journal of China

基　　金：云南省教育厅科学研究基金(2023Y0642);昆明医科大学第二附属医院院内科技计划项目(2021yk019);中央高水平医院临床研究基金(2022-PUMCH-A-006)

摘　　要：目的在类中性粒细胞模型中,探讨尿激酶型纤溶酶原激活物受体(PLAUR)基因对中性粒细胞活化及凋亡的影响。方法采用体外培养的人急性髓系粒细胞白血病细胞株HL60作为研究对象,全反式维甲酸(ATRA)诱导HL60细胞分化为类中性粒细胞。构建干扰人PLAUR基因的慢病毒载体,转染入类中性粒细胞(siRNA组),并设置磷酸盐缓冲液(PBS)组(未转染)和空白对照(NC组,转染空白质粒)作为对照(n=3)。饥饿培养、加入白细胞介素-17干预后,流式细胞术检测细胞膜CD11b表达,以及酶联免疫吸附法检测细胞培养液上清中髓过氧化物(MPO)、中性粒细胞胞外诱捕网(NETs)水平,了解类中性粒细胞活化情况。Annexin V/PI染色流式细胞术检测细胞凋亡,并用Western蛋白印迹法检测凋亡相关蛋白caspase-3、bax、bcl-2表达。结果流式细胞术检测siRNA组HL60细胞细胞膜CD11b表达(32.37±8.17)较另两组下降(PBS组46.27±1.54,NC组53.07±8.14)(P<0.05)。siRNA组细胞培养液上清中MPO和NETs水平(33.37±1.11、57.69±3.03)也均较PBS组(41.64±2.20、77.60±4.33)和NC组(40.84±5.11、76.15±2.10)下降,差异均有统计学意义(均P<0.05)。流式细胞术检测siRNA组凋亡率(20.42%±2.45%)较PBS组(11.91%±2.23%)和NC组(11.13%±2.56%)升高(P<0.05)。siRNA组caspase-3和bax蛋白表达(0.84±0.05、0.83±0.04)较PBS组(0.68±0.02、0.63±0.08)和NC组(0.71±0.01、0.66±0.10)高,差异有统计学意义(P<0.05);抗凋亡蛋白bcl-2表达量减少(siRNA组:0.38±0.02,PBS组:0.73±0.05,NC组:0.69±0.06)(P<0.05)。结论PLAUR可促进类中性粒细胞活化,抑制其凋亡。Objective To investigate the effect of plasminogen activator urokinase receptor(PLAUR)gene on neutrophil activation and apoptosis in neutrophil-like cell model.Methods Human acute myeloid leukemia cell line HL60 was cultured in vitro and induced to differentiate into neutrophil-like cells by all-trans retinoic acid(ATRA).Lentiviral vectors interfering with human PLAUR gene was constructed and transfected into neutrophil-like cells(siRNA group).The phosphate buffer saline(PBS)group(untransfected neutrophil-like cells)and normal blank control group(NC group)(neutrophil-like cells transfected with blank plasmid)were used as controls(n=3).After starvation culture and addition of interleukin-17 afterwards in these 3 groups,the expression of CD11b on the cell membrane was detected by flow cytometry,and the levels of myeloperoxide(MPO)and extracellular neutrophil traps(NETs)in the supernatant were detected by enzyme-linked immunosorbent assay(ELISA)to investigate the activation of neutrophil-like cells.The apoptosis was detected by flow cytometry with annexin V/propidium iodide(PI)double staining and the expressions of apoptosis-related proteins caspase-3,bax and bcl-2 were detected by Western blotting.Results The expression of CD11b in siRNA group(32.37±8.17)was lower than that in PBS group(46.27±1.54)and NC group(53.07±8.14)(P<0.05)by flow cytometry.The levels of MPO and NETs(33.37±1.11,57.69±3.03)in the supernatant of siRNA group were significantly lower than those in PBS group(41.64±2.20,77.60±4.33)and NC group(40.84±5.11,76.15±2.10)(P<0.05).Flow cytometry with annexin V/PI showed that the expression of apoptosis in siRNA group(20.42%±2.45%)was significantly higher than that in PBS group(11.91%±2.23%)and NC group(11.13%±2.56%)(P<0.05).The relative expression of caspase-3 protein and bax protein(0.84±0.05,0.83±0.04)in siRNA group was significantly higher than that in PBS group(0.68±0.02,0.63±0.08)and NC group(0.71±0.01,0.66±0.10)(P<0.05),and the relative expression of anti-apoptosis protein bcl-2 de

关 键 词：皮肌炎 尿激酶型纤溶酶原激活物受体基因 中性粒细胞活化 凋亡 间质性肺病 

分 类 号：R593.26[医药卫生—内科学]