巨噬细胞通过下调miR-20a诱发凋亡促进结核菌清除的机制

目的 探讨上调或下调巨噬细胞中miR-20a对结核菌诱导的细胞凋亡以及细菌清除的影响，鉴定结核免疫干扰新靶点。方法 构建表达miR-20a-5p、miR-20a-5p-inh且含绿色荧光蛋白（GFP）的慢病毒载体，用无血清转染试剂稀释，在5 μg/mL聚凝胺存在下以感染复数（MOI）为10转染人单核细胞株THP-1细胞3 h，72 h后通过流式细胞仪筛选出GFP+ 细胞，佛波脂（PMA）刺激24 h后分化为巨噬细胞；结核分枝杆菌灭活菌（H37Ra）或过氧化氢（H2O2）处理细胞后, 进行荧光素PE标记的膜联蛋白（Annexin）V和7-氨基放线菌素D（7AAD）染色15 min，流式细胞术检测Annexin V（+）和Annexin V（+）7AAD（+）细胞早晚期凋亡情况,方差分析（ANOVA）和多重比较试验（Newman-Keuls）分析细胞间凋亡率的差异；H37Ra 以MOI为10感染巨噬细胞30 min，磷酸盐缓冲液（PBS）洗3次后放入CO2培养箱，3 d后收集裂解物并接种到含10％OADC增菌液的7H11琼脂平板上，37 ℃培养箱培养3~4周后使用标准程序重复三次计算细菌菌落数。结果 成功构建miR-20a-5p、miR-20a-5p-inh慢病毒载体以及稳定促进或抑制miR-20a表达的细胞株；流式细胞术检测显示：在未刺激情况下细胞早、晚期凋亡的差异不明显，H37Ra或H2O2刺激后，细胞早、晚期凋亡率发生明显变化，以晚期凋亡率显著增加为主；方差分析和多重比较试验提示：在未刺激情况下过表达或抑制miR-20a，细胞凋亡率无显著差异，H37Ra或H2O2刺激后在miR-20a过表达时细胞凋亡率无明显变化，抑制miR-20a的表达细胞凋亡率显著增加；H37Ra感染巨噬细胞后收集细胞裂解物并接种到平板中培养，miR-20a过表达的细胞内结核菌存活率较高，抑制miR-20a表达结核菌的存活率下降。结论 结核菌感染巨噬细胞后，宿主通过下调miR-20a诱发细胞凋亡进而清除结核菌。

Down-regulation of miR-20a triggers macrophages cell apoptosis to facilitate mycobacterial clearance

Objective To investigate the effect of up-regulation or down-regulation of miR-20a on the macrophage apoptosis induced by M. tuberculosis and bacterial eradication, and to identify the new target for tuberculosis immune interference. Methods Lentiviral vector expressing miR-20a-5p, miR-20a-5p-inh and containing green fluorescent protein (GFP) was constructed, then the lentivirus were diluted in serum free OptiMEM and transfected into human mononuclear cell line (THP-1) cell at a multiplicity of infection (MOI) of 10 for 3 h in the presence of 5 μg/mL polybrene, from which the GFP+ cells were isolated with fluorescence activated cell sorting (FACS) after 72 h of infection in serum-containing medium for further analysis and differentiate into macrophages in the presence of phorbol 12-myristate 13-acetate (PMA, 20 ng/mL) for 24 hours; the cells were stimulated by the live attenuated strain H37Ra or treated with hydrogen peroxide (H2O2) and incubated with annexin V-PE and 7-amino actinomycin D (7AAD) for 15 min, the annexin V(+) and annexin V(+) 7AAD (+) cells were defined as the number of apoptotic cells, determined by flow cytometric analysis. ANOVA/Newman-Keuls multiple comparison test was used to compare the difference of apoptosis rates among various groups; cells were infected by H37Ra for 30 min at a MOI of 10 and washed 3 times with PBS, and 3 d later, the cell lysates were collected, serial dilutions of lysates were plated on 7H11 agar plates supplemented with 10% OADC and incubated at 37 ℃ for 3–4 weeks, the number of bacterial colonies was counted in triplicate with standard procedure. Results The miR-20a-5p, miR-20a-5p-inh lentiviral vectors were successfully constructed, and the THP-1 cell line that miR-20a gene stably increased or suppressed were established; flow cytometry analysis showed that there was no obvious difference between early and late apoptosis in the absence of stimulation, the cell apoptosis changed obviously after infected by H37Ra or H2O2, with the increase of late apoptosis significantly; The one-way analysis of variance (ANOVA)/Newman-Keuls multiple comparison test was used for statistical analyses to indicate that there was no significant difference in the apoptotic rate of miR-20a over-expressing or inhibit-expressing without stimulation, then we found the apoptosis rate was significantly increased when miR-20a expression was inhibited after H37Ra or H2O2 stimulation although there was also no significant change in the rate of apoptosis in miR-20a-overexpressed cells; the cells were infected by H37Ra, then the lysates were collected and inoculated into the plate, the results showed than the survival of Mtb was augmented in the presence of miR-20a over-expressing cells, and compromised in cells treated with miR-20a-5p-inh. Conclusions The host can clear the Mycobacterium tuberculosis by down-regulating miR-20a-induced apoptosis of macrophages.