HRZ三联抗结核药/TGF-β1 siRNA纳米脂质体对体外BCG感染的人巨噬细胞中Ag85A及TGF-β1表达的影响

Effects of HRZ triple antituberculosis drug/TGF-β1 siRNA nanoliposomes on the expression of Ag85A and TGF-β1 in human BCG-infected human macrophages

目的:探讨异烟肼、利福平、吡嗪酰胺(HRZ)三联抗结核药/表皮转化生长因子(TGF)-β1 si RNA纳米脂质体对体外BCG感染的人巨噬细胞中Ag85A及TGF-β1表达的影响。方法:体外培养人单核细胞株THP-1诱导分化成巨噬细胞,分为4组,空白组为单纯巨噬细胞培养;模型组为巨噬细胞与卡介苗(bacillus calmetteguerin,BCG)以1∶5的比例共培养3h,制备BCG感染的巨噬细胞模型;对照组为BCG感染的巨噬细胞与HRZ三联抗结核药纳米脂质体共培养;实验组为BCG感染的巨噬细胞与三种不同浓度的HRZ三联抗结核药/TGF-β1 si RNA纳米脂质体(C1组为35mg/ml,C2组为40mg/ml,C3组为50mg/ml)共培养。采用RT-PCR检测各组人巨噬细胞中Ag85A及TGF-β1的mRNA表达量,Western-blot方法检测Ag85A及TGF-β1的蛋白表达量。结果:与空白组相比,模型组Ag85A和TGF-β1的m RNA及蛋白量表达明显上调(P<0.05);对照组中Ag85A的mRNA及蛋白量和TGF-β1的mRNA水平与模型组相比明显下调(P<0.05);实验组3种不同浓度(C1组、C2组、C3组)HRZ三联抗结核药/TGF-β1 si RNA纳米脂质体处理后,Ag85A和TGF-β1的mRNA及蛋白表达量与模型组和对照组相比进一步下调(P<0.05),随着纳米脂质体浓度的增加,Ag85A和TGF-β1 m RNA及蛋白表达量有明显下降趋势,C1组与C3组相比Ag85A m RNA及蛋白表达量差异有统计学意义(P<0.05);C1组与C2组、C3组相比TGF-β1 mRNA及蛋白量表达差异有统计学意义(P<0.05);C2组与C3组相比Ag85A m RNA和TGF-β1蛋白表达量差异有统计学意义(P<0.05)。结论:HRZ三联抗结核药/TGF-β1 si RNA纳米脂质体对体外BCG感染的人巨噬细胞具有明显的下调Ag85A基因表达作用及TGF-β1基因沉默作用。

Objectives： To investigate the effects of isoniazid/rifampicin/pyrazinamide （HRZ） triple anti- tuberculosis/epidermal transforming growth factor （TGF）-β1 siRNA nanoliposomes on the expression of Ag85A and TGF-β1 in human macrophages in vitro. Methods： The human mononuclear cell strain THP-1 was induced to differentiate into macrophages in vitro. The experiment was divided into 4 groups. The blank group was cultured as macrophage alone. In the model group, macrophage and bacillus calmette-guerin（BCG）were cocuhured at the ratio of 1：5 for 4h to prepare a BCG-infected macrophage model. In the control group, BCG-infected macrophages and HRZ triple antituberculosis nanoliposomes were cocultured. In the experimental group, BCG-infected macrophages and HRZ triple anti-tuberculosis drug/TGF-β1 siRNA nanoliposome（C1 35mg/ml, C2 40mg/ml, and C3 50mg/ml） were cococultured. The mRNA expression levels of Ag85A and TGF-β1 in human macrophages were detected by RT-PCR. The protein expression levels of Ag85A and TGF-β1 were detected by western-blot method. Results： RT-PCR and western-blot results showed that the mRNA and protein levels of Ag85A and TGF-β1 were significantly up-regulated in the model group （BCG-infected macrophage model） compared with the blank group （macrophage culture alone）（P〈 0.05）. The mRNA and protein expressions of Ag85A and TGF-β1 in the control group（cocuhured with BCG- infected macrophages and HRZ triple anti-tuberculosis nano-liposomes） were significantly down-regulated compared with the model group（P〈0.05）; Compared with the model group and the control group, the mRNAs and protein expressions of Ag85A and TGF-β1 treated with HRZ triple antituberculosis drugs/TGF-β1 siRNA nanoliposomes in three different concentrations（C1 group, C2 group and C3 group） in the experimental group was further down-regulated （P〈0.05）. With the increase of nano-liposome concentration （35mg/ml, 40mg/ml, 50mg/ml）, the expressions of TGF-β1 mRNA and protein decreased significantly （the difference between C1 group and C2 group and C3 group was statistically significant, P〈0.05）. Conclusions： The self-developed HRZ triple anti-tuberculosis drug/TGF-β1 siRNA nano-liposome has obvious down-regulation of Ag85A gene expression and TGF-β1 gene silencing.