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

Abstract:

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 (MDR-group) 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 (R_PMN and S_PMN) and MTB (R_MTB and S_MTB) were co-cultured in vitro, and 6 groups of detection specimens were obtained, including R_PMN, R_PMN+R_MTB, R_PMN+S_MTB, S_PMN, S_PMN+R_MTB, S_PMN+S_MTB. 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 R_PMN group without MTB antigen stimulation ((30.67±3.03) %) was significantly lower than that of the S_PMN group ((36.54±2.49) %), but higher than that of the R_PMN+R_MTB group ((21.74±3.35) %), with statistical significance (t=4.728 and 6.254, respectively, P values were 0.000); R_PMN+R_MTB group was significantly lower than S_PMN+S_MTB group ((50.84±3.38) %) and R_PMN+S_MTB group ((34.66±3.05) %), and the differences were statistically significant (t=16.699 and 9.018, respectively, P values were 0.000).The S_PMN+S_MTB group was significantly higher than the S_PMN+R_MTB group ((33.56±2.94) %) and the S_PMN group, the differences were statistically significant (t=12.200 and 8.974, respectively, P values were 0.000).TLR2 ((24.94±1.77) ng/ml) and Caspase-3 (6.77±0.73) levels of PMN in S_PMN group without MTB antigen stimulation were significantly higher than those in R_PMN group ((13.46±1.67) ng/ml and 4.85±0.91), with statistical significance (t=14.931 and 5.224, respectively, P values were 0.000); TLR2 ((28.32±3.00) ng/ml) and Caspase-3 (6.33±0.93) levels of PMN in S_PMN+S_MTB group were significantly higher than those in R_PMN+R_MTB group ((15.25±2.10) ng/ml and 4.95±1.07), with statistical significance (t=11.297 and 3.063, P=0.000 and 0.007, respectively).TLR2 level in S_PMN group was significantly higher than that in S_PMN+R_MTB group ((22.23±1.90) ng/ml), but significantly lower than that in S_PMN+S_MTB group (LSD-test,Ps<0.05,respectively). Conclusion The apoptosis rate, degree of caspase-3 activation and TLR2 level of PMN stimulated by MDR-TB were lower than those of PMN without MTB antigen stimulation,suggesting that PMN of MDR-TB patients may have apoptosis inhibition related to the down-regulation of TLR2 level and the decrease of caspase-3 activation.

Key words: Mycobacterium tuberculosis, Tuberculosis, multidrug-resistant, Human neutrophil, Antimycobacterial activity, Apoptosis