耐多药肺结核患者外周血中性粒细胞抗结核活性的初步研究  

The study on the anti-tuberculosis activity of polymorphonuclear cell from peripheral blood of patients with multidrugresistant pulmonary tuberculosis

作　　者：刘婷 王亮[3] 杨梅 LIU Ting;WANG Liang;YANG Mei(Department of Clinical Laboratory,Bao’an Hospital of Traditional Chinese Medicine(Group),Shenzhen 518100,China)

机构地区：[1]深圳市宝安中医院(集团)检验科,518100 [2]深圳市宝安区慢性病防治院检验科 [3]香港大学深圳医院介入诊疗科 [4]深圳市宝安区人民医院感染科

出　　处：《中国防痨杂志》2021年第4期352-356,共5页Chinese Journal of Antituberculosis

基　　金：深圳市科技计划基础研究项目(JCYJ20160427092554740);深圳市宝安区科技计划基础研究项目(2018JD115)

摘　　要：目的探讨耐多药结核分枝杆菌(Mycobacterium tuberculosis,MTB)的耐药特性是否与其减弱人外周血多形核白细胞(polymorphonuclear leukocyte,PMN)的凋亡能力,从而逃避机体免疫杀伤有关。方法搜集2017年5月至2018年9月就诊于深圳市宝安区慢性病防治院并确诊的10例耐多药肺结核患者(耐多药组)和10例药物敏感肺结核患者(非耐药组),分别获取其MTB临床分离株并冻存,菌株均经比例法药物敏感性试验确证。分别抽取患者外周血6ml,分离PMN(两组分别为RPMN和SPMN),同时制备MTB抗原(两组分别为RMTB和SMTB)。将分离出的PMN与MTB体外共培养后,获得RPMN、RPMN+RMTB、RPMN+SMTB、SPMN、SPMN+RMTB、SPMN+SMTB共6组检测标本。应用荧光倒置显微镜、酶联免疫吸附试验、分光光度法观察PMN的凋亡率,以及检测PMN中Toll样受体2(Toll-like receptor 2,TLR2)水平和半胱氨酸蛋白酶-3(Caspase-3)活化程度。结果未经MTB抗原刺激的RPMN组的凋亡率[(30.67±3.03)%]明显低于SPMN组[(36.54±2.49)%],但高于RPMN+RMTB组[(21.74±3.35)%],差异均有统计学意义(t值分别为4.728和6.254,P值均为0.000);而RPMN+RMTB组明显低于SPMN+SMTB组[(50.84±3.38)%],也明显低于RPMN+SMTB组[(34.66±3.05)%],差异均有统计学意义(t值分别为16.699和9.018,P值均为0.000);SPMN+SMTB组明显高于SPMN+RMTB组[(33.56±2.94)%],也明显高于SPMN组,差异均有统计学意义(t值分别为12.200和8.974,P值均为0.000)。未经MTB抗原刺激的SPMN组PMN的TLR2[(24.94±1.77)ng/ml]及Caspase-3(6.77±0.73)水平均明显高于RPMN组[分别为(13.46±1.67)ng/ml和4.85±0.91],差异均有统计学意义(t值分别为14.931和5.224,P值均=0.000);而SPMN+SMTB组PMN的TLR2[(28.32±3.00)ng/ml]及Caspase-3(6.33±0.93)水平均明显高于RPMN+RMTB组[分别为(15.25±2.10)ng/ml和4.95±1.07)],差异均有统计学意义(t值分别为11.297和3.063,P值分别为0.000和0.007);SPMN组的TLR2水平明显高于SPMN+RMTB组[(22.23±1.90)ng/ml],但明显低于SPMN+SMTB�Objective To investigate whether the drug resistance of Mycobacterium tuberculosis(MTB)is related to the impaired ability to induce apoptosis of human peripheral blood polymorphonuclear cell(PMN),thereby avoiding immune killing. Methods A total of 10 MDR-TB(multidrug-resistant tuberculosis)patients(MDRgroup)and 10 drug-sensitive tuberculosis patients(non-drug-resistant group)who were diagnosed by Bao’an Hospital of Chronic Diseases Control and Prevention,Shenzhen from May 2017 to September 2018 were collected.MTB clinical isolates were obtained and frozen.All the strains were confirmed by drug sensitivity test using proportional method.6 ml of peripheral blood was extracted from patients and PMN was isolated,meanwhile,MTB antigen was prepared.The isolated PMN(RPMNand SPMN)and MTB(RMTBand SMTB)were co-cultured in vitro,and6 groups of detection specimens were obtained,including RPMN,RPMN +RMTB,RPMN +SMTB,SPMN,SPMN +RMTB,SPMN+SMTB.The apoptotic rate of PMN was observed by fluorescence inversion microscopy.The level of Toll-like receptor 2(TLR2)and the activation degree of Caspase-3 in PMN were detected by ELISA and spectrophotometry,respectively. Results The apoptosis rate of the RPMNgroup without MTB antigen stimulation((30.67±3.03)%)was significantly lower than that of the SPMNgroup((36.54±2.49)%),but higher than that of the RPMN+RMTB group((21.74±3.35)%),with statistical significance(t=4.728 and 6.254,respectively,Pvalues were 0.000);RPMN+RMTB group was significantly lower than SPMN +SMTB group((50.84±3.38)%)and RPMN +SMTB group((34.66±3.05)%),and the differences were statistically significant(t=16.699 and 9.018,respectively,Pvalues were 0.000).The SPMN+SMTBgroup was significantly higher than the SPMN+RMTBgroup((33.56±2.94)%)and the SPMN group,the differences were statistically significant(t=12.200 and 8.974,respectively,P values were0.000).TLR2((24.94±1.77)ng/ml)and Caspase-3(6.77±0.73)levels of PMN in SPMN group without MTB antigen stimulation were significantly higher than those in RPMN group((13.46�

关 键 词：分枝杆菌 结核 结核 抗多种药物性 人中性粒细胞 抗结核作用 细胞凋亡 

分 类 号：R521[医药卫生—内科学]