肺泡灌洗液中miR-30a-5p、CCL16表达与肺结核患者结核杆菌耐药性的关系

The relationship between the expression of miR-30a-5p and CCL16 in bronchoalveolar lavage fluid and the drug resistance of Mycobacterium tuberculosis in patients with pulmonary tuberculosis

目的 探讨耐药肺结核患者支气管肺泡灌洗液中微小RNA-30a-5p(microRNA-30a-5p, miR-30a-5p)和C-C基序趋化因子配体16(C-C motif chemokine ligand 16, CCL16)表达水平变化,为结核病防治提供理论基础。方法 选取2018年2月1日-2021年6月30日在我院治疗的活动性肺结核患者182例作为研究对象(观察组),并根据是否对结核杆菌耐药情况将其分为耐药组和非耐药组,选择同期90例非肺结核的健康人(因胸痛经纤维支气管镜检查排除肺结核)作为对照组。采用实时荧光定量PCR法检测支气管肺泡灌洗液中miR-30a-5p表达水平,采用ELISA法检测CCL16水平,采用流式细胞分析法检测外周血T细胞亚群分布,采用Pearson分析法分析耐药肺结核患者支气管肺泡灌洗液中miR-30a-5p与CCL16表达水平相关性,采用Logistic逐步回归分析法分析肺结核患者结核杆菌耐药性的影响因素。结果 非耐药组与耐药组CD3^(+)T细胞、CD4^(+)T细胞水平及CD4/CD8比值均显著低于对照组,CD8^(+)T细胞水平显著高于对照组(P均<0.05);耐药组CD3^(+)T细胞、CD4^(+)T细胞水平及CD4/CD8比值均显著低于非耐药组,CD8^(+)T细胞水平显著高于非耐药组(P均<0.05)。非耐药组与耐药组支气管肺泡灌洗液中miR-30a-5p表达水平均显著低于对照组,CCL16水平均显著高于对照组(P均<0.05);耐药组肺泡灌洗液中miR-30a-5p表达水平显著低于非耐药组,CCL16水平显著高于非耐药组(P均<0.05)。有吸烟史、广泛耐药的肺结核患者肺泡灌洗液中miR-30a-5p表达水平显著低于无吸烟史、耐多药的肺结核患者,CCL16水平显著高于无吸烟史、耐多药的肺结核患者(P均<0.05)。Pearson相关性分析显示,耐药肺结核患者肺泡灌洗液中miR-30a-5p与CCL16表达水平呈显著负相关(r=-0.556,P <0.001);Logistics多因素回归分析显示,有吸烟史、肺部空洞、miR-30a-5p <0.72、CCL16≥5.34 pg/ml为影响肺结核患者耐药性的危险因素(P均<0.05)。结论 耐药肺结核患者支气管肺泡灌洗液中miR-30a-5p表达水平降低,CCL16水平升高,检测miR-30a-5p、CCL16水平可反映机体感染结核杆菌后机体发生的免疫应答情况,为耐药肺结核的治疗提供参考。

Objective To investigate the changes in the expression levels of microRNA-30a-5p(miR-30a-5p) and C-C motif chemokine ligand 16(CCL16) in bronchoalveolar lavage fluid of patients with drug resistant pulmonary tuberculosis, so as to provide a theoretical basis for the prevention and treatment of tuberculosis. Methods One hundred and eighty-two patients with active pulmonary tuberculosis who were treated in our hospital from February 1, 2018 to June 30, 2021 were selected as the study subjects(observation group), they were divided into drug resistant group and non drug resistant group according to whether they were drug resistant to Mycobacterium tuberculosis, and 90 healthy people without pulmonary tuberculosis(excluding pulmonary tuberculosis due to chest pain through fiberoptic bronchoscopy) in the same period were selected as control group. The expression level of miR-30a-5p in bronchoalveolar lavage fluid was detected by real-time quantitative PCR, and the level of CCL16 was detected by ELISA, distribution of T cell subsets in peripheral blood were detected by flow cytometry, the correlation between miR-30a-5p and CCL16 expression in bronchoalveolar lavage fluid of drug resistant pulmonary tuberculosis patients was by Pearson method, the influencing factors of drug resistance of Mycobacterium tuberculosis in patients with pulmonary tuberculosis were analyzed by Logistic regression. Results The levels of CD3^(+)T cells, CD4^(+)T cells and the ratio of CD4/CD8 in non drug resistant group and drug resistant group were significantly lower than that of control group, and the level of CD8^(+)T cells were significantly higher than that of control group(P <0.05). The levels of CD3+T cells, CD4^(+)T cells and the ratio of CD4/CD8 in drug resistant group were significantly lower than that of non drug resistant group, and the level of CD8^(+)T cells were significantly higher than that of non drug resistant group(P <0.05). The expression level of miR-30a-5p in the bronchoalveolar lavage fluid of non drug resistant group and drug resistant group were significantly lower than that of control group, and the level of CCL16 was significantly higher than that of control group(P <0.05). The expression level of miR-30a-5p in the bronchoalveolar lavage fluid of drug resistant group was significantly lower than that of the non drug resistant group,and the level of CCL16 was significantly higher than that of non drug resistant group(P <0.05). The expression level of miR-30a-5p in bronchoalveolar lavage fluid of pulmonary tuberculosis patients with smoking history, extensively drug-resistant tuberculosis was significantly lower than that of pulmonary tuberculosis with no smoking history, multi-drug resistant tuberculosis, and the level of CCL16was significantly higher than that of pulmonary tuberculosis with no smoking history, multi-drug resistant tuberculosis(P <0.05).Pearson correlation analysis showed that there was a significant negative correlation between miR-30a-5p and CCL16 expression in bronchoalveolar lavage fluid of drug resistant pulmonary tuberculosis patients(r=-0.556, P <0.05). Logistics multi-factor regression analysis showed that smoking history, pulmonary holes, miR-30a-5p < 0.72, CCL16 ≥ 5.34 pg/ml were risk factors affecting drug resistance in pulmonary tuberculosis patients(P <0.05). Conclusions The expression level of miR-30a-5p in bronchoalveolar lavage fluid of drug resistant pulmonary tuberculosis patients decreased, and the level of CCL16 increased, the detection of miR-30a-5p and CCL16 levels can reflect the immune response of the body after infection with Mycobacterium tuberculosis, and provide reference for the treatment of drug resistant pulmonary tuberculosis.