半乳糖凝集素-1对结核分枝杆菌感染巨噬细胞的调节作用及其临床价值

Regulation and clinical value of galectin-I on macrophages infected with Mycobacterium tuberculosis

目的分析半乳糖凝集素-1（galectin-1，Gal1）对结核分枝杆菌（MTB）感染巨噬细胞的调节作用，并探讨其血清含量与结核病的临床相关性。方法选取2014年612月于同济大学附属上海市肺科医院门诊就诊的活动性结核病患者40例作为病例组；选取同时期结核菌素皮肤试验（tuberculinskintest，TST）阳性潜伏感染者20例作为观察组；参照观察组患者的年龄和性别构成，按照1：1的比例，选择同期体检TST阴性健康者20名作为对照组。研究对象各采集外周血5ml，采用El。ISA检测其血清Gal-1含量；分别采用蛋白质免疫印迹（Westernblot）和ELISA检测MTB感染人单核巨噬细胞（THP1）后，Gal-1的表达及分泌情况；应用流式细胞术检测外源性Gal1（设置添加10、50ng／ml的GaLl组和空白对照组）对人单核巨噬细胞（THP1）吞噬表达绿色荧光蛋白（greenfluorescentprotein，GFP）的MTB（MTB-GFP）的影响；应用胞内存活实验检测外源性Gal—l对巨噬细胞清除MTB能力的作用。结果血清Gal-1含量病例组为（28．30±1．17）ng／ml，高于对照组的（21．28±1．74）ng／ml和观察组的（22．06±1．25）ng／ml，差异有统计学意义（F=5．50，P=0．006）。MTB感染THP一1细胞2h后，Gal1表达量增加。感染复数（multiplicityofinfection，MOI）值为1和5时，细胞培养液上清液中Gal-1含量分别为（391．27±34．45）pg／ml和（751．54±148．93）pg／ml。添加10、50ng／ml Gal-1组与空白对照组MTB-GFP阳性THP-1细胞的比率分别为（6．77±0．58）％、（6．93±0．62）％和（7．64±0．74）％，三组比较差异无统计学意义（F=1．53，P=0．291）；MTDGFP阳性THP-I细胞的平均荧光强度分别为321．67±5．03、318．00±13．89、342．67±13．65，三组比较差异无统计学意义（F=3．94，p=0．081）。感染24h后，添加10ng／ml和50ng／mlGal-1组胞内克隆形成单位数[M（Q1～Q3）]分别为3250（2825～6225）、3250（1850～4950），与空白对照组[9250（8525～111550）]比较，差异有统计学意义（U=0．00，P=0．029；U=0．00，p=0．028）；感染72h后，分别为19500（17500～21500）、12000（11250～15750），与空白对照组E38500（31750～44500）]比较，差异有统计学意义（U=0．00，P=0．029；U=0．00，P=0．029）。结论活动性结核病患者血清中Gal1表达水平异常升高，胞外Gabl可增强巨噬细胞对MTB的杀伤功能。因此，Oal-1具有一定的结核病辅助诊断价值，并可能通过影响巨噬细胞功能参与结核病发病过程。

Objective To analyze the regulatory role of galectin-1 （Gal-1） in the process of macrophages in- fected with Mycobacterium tuberculosis （MTB） and to evaluate the correlation of its serum abundance with pulmonary tuberculosis. Methods From Shanghai Pulmonary Hospital affiliated to Tongii University between June 2014 and December 2014, 40 outpatients with active pulmonary tuberculosis were enrolled as patient group, 20 latent infection individuals with positive tuberculin skin test （TST） were in experimental group, and 20 healthy individuals were selected as control group; 5 ml peripheral biood were collected from each person and enzyme-linked immunosorbent assay （ELISA） was used to test Gal-1 in serum. The expression and secretion of Gal 1 after THP-1 infected with MTB were detected by Western blot and EI.ISA. Furthermore, the effects of exogenous Gal-1 （divided into Gal-1-treated group （10 ng/ml and 50 ng/ml） and untreated group） on the uptake of MTB stably transfected with green fluorescent protein （MTB-GFP） in THP-1 and the clearance of MTB in macrophages were detected by flow cytome- try and intracellular survival test, respectively. Results The abundance of Gal-1 in serum was （28.30±1.17） ng/ml in patient group, which was higher than those of experimental group （（21.28 ± 1.74） ng/ml） and control group （（22.06±1.25） ng/ml）, and the difference was statistically significant （F=5.50, P=0. 006）. The expression of Gal-1 increased 2 hours after THP-1 being infected with MTB. Gal-1 in supernatant of cell culture medium were （391.27±34.45） pg/ml and （751.54±148.93） pg/ml when multiplicity of infection （MOI） at 1 and 5, respective- ly. In Gal 1-treated group （10 and 50 ng/ml） and untreated group, the percentages of THP-1 cells with MTP-GFP positive were （6.77±0. 58）%, （6.93±0.62）% and （7.64±0.74） %, respectively, differences among these groups were not statistically significant （F= 1.53, P= 0. 291）, and neither was the mean fluorescent intensity （MFI） （F=3.94, P=0. 081）, which were 321.67±5.03, 318.00±13.89 and 342.67±13.65, respectively. The colony-forming unit （CFU, M （Q1 Qa ）） 24 hours post-infection of Gal-l-treated group （10 ng/ml： 3250 （2825- 6225）, 50 ng/ml; 3250 （1850--4950）） were statistically significant from that of untreated group （9250 （8525- 111 550）） （U=0.00, P=0. 029; U=0. 00, P=0. 028）, as well as the CFU 72 hours post-infection （the CFU were 38 500 （31 750--44 500）, 19 500 （17 500--21 500） and 12 000 （11 250--15 750） in untreated and Gal 1-treated （10 and 50 ng/ml） groups, respectively （U=0. 00, P=0. 029; U=0. 00, P〈0. 029））. Conclusion Gal-1 abber- antly increases in serum of active TB patients, and extracellular Gab1 enhances the killing activity of macrophages against MTB. Therefore, Gal-1 could serve as a potential biomarker for the diagnosis of TB. It may be involved in the pathogenesis of TB by modulating the function of macrophages.