LI Peng,CUI Jia,WANG Liqing,et al.ASAP1 involvement of the phagocytosis of Mycobacterium tuberculosis in macrophages[J].Journal of Nanjing Agricultural University,2018,41(3):481-488.[doi:10.7685/jnau.201801042]





ASAP1 involvement of the phagocytosis of Mycobacterium tuberculosis in macrophages
李鹏 崔佳 王丽清 吴长新
山西大学生物医学研究院, 山西 太原 030006
LI Peng CUI Jia WANG Liqing WU Changxin
Institutes of Biomedical Sciences, Shanxi University, Taiyuan 030006, China
ASAP1macrophagesMycobacterium tuberculosis(Mtb)phagocytosis
[目的]结核病是全球最重要的人畜共患传染病之一,Arf核糖激化因子GTP酶活化蛋白(ASAP1)与结核病易感性相关。本文旨在明确在细胞水平ASAP1表达与结核分枝杆菌感染的关系,探究ASAP1是否参与巨噬细胞对Mycobacterium tuberculosisMtb)的吞噬以及了解该蛋白表达对巨噬细胞吞噬结核分枝杆菌的影响。[方法]以人源单核细胞建立的不同ASAP1表达水平的巨噬细胞作为模型,通过感染结核分枝杆菌开展了一系列体外试验,使用BCG和Mtb H37Ra菌株感染THP-1分化的巨噬细胞,Western blot以及荧光定量PCR检测感染后ASAP1的表达;建立不同ASAP1表达水平的巨噬细胞,进一步研究ASAP1对巨噬细胞吞噬Mtb H37Ra的调节作用;利用Mtb H37Ra感染不同表达ASAP1的巨噬细胞,通过细菌菌落计数及细胞免疫荧光试验,检测ASAP1表达水平与巨噬细胞吞噬Mtb H37Ra的关系。[结果]Mtb H37Ra感染诱导ASAP1表达上调,BCG感染不能诱导ASAP1表达上调,说明ASAP1参与了巨噬细胞对Mtb H37Ra的吞噬作用;利用Mtb H37Ra感染不同ASAP1表达水平的巨噬细胞,证实ASAP1低水平表达的巨噬细胞对Mtb H37Ra吞噬能力减弱,高水平表达的巨噬细胞对Mtb H37Ra的吞噬能力增强,ASAP1表达水平与巨噬细胞对Mtb H37Ra的吞噬能力呈正相关。[结论]Mtb感染能诱导ASAP1高水平表达,ASAP1参与巨噬细胞对Mtb吞噬,并通过该蛋白表达对巨噬细胞吞噬结核分枝杆菌进行调控,ASAP1的超高水平表达可诱导巨噬细胞对Mtb过多地吞噬。
[Objectives] Tuberculosis(TB)is one of the most important zoonosis all over the world. ASAP1 is associated with the susceptibility to TB in humans. The purpose of this research is to investigate the relationship between ASAP1 expression and Mtb infection, prove whether ASAP1 involves the phagocytosis of Mycobacterium tuberculosis (Mtb)in macrophages, and understand how ASAP1 affects the phagocytosis of Mtb in macrophages. [Methods] By using cell model, a THP-1 derived macrophage model expressing different levels of ASAP1 was generated, then a serials experiments were performed for analysis of affection of ASAP1 on phagocytosis of Mtb after infection of Mtb or BCG in vitro. BCG and Mtb H37Ra were used to infect macrophages, and ASAP1 expression was tested by using Western blotting and Real-time qPCR. ASAP1 specific siRNA was used to generate ASAP1 knockdown macrophages, ASAP1 expression lentiviral system was constructed to transduce THP-1 cell, and then it was made to differentiate into ASAP1 overexpression macrophages. Mtb H37Ra was used to infect macrophages expressing three different ASAP1 expression levels including knockdown, overexpression and normal expression of macrophages which were directly derived from THP-1 cell line, and then the intracellular Mtb number post-infection was counted by using colony forming unit(CFU)counting assays and image acquisition technique. [Results] This research first demonstrated that Mtb H37Ra infection significantly provoked higher ASAP1 expression in macrophages derived from THP-1 compared with BCG infection and non-infection control. ASAP1 overexpression macrophages(P<0.01)and knockdown macrophages(P<0.01)were generated to establish a macrophage cellular model for investigation into the affection of ASAP1 on phagocytosis of Mtb in macrophages. The CFU assays showed that ASAP1 overexpression macrophages loaded more bacteria(P<0.001), and ASAP1 knockdown macrophages loaded less bacteria compared with macrophages directly derived from wild type THP1 as a control(P<0.001). [Conclusions] Mtb infection provokes higher ASAP1 expression in macrophages, and a macrophage cellular model has been established for investigation into the mechanisms underling ASAP1 expression affecting on phagocytosis of Mtb in macrophages. Results also have shown ASAP1 expression has positive correlation with phagocytosis of Mtb, and overexpression of ASAP1 may introduce redundant phagocytosis of Mtb in macrophages, which may affect humans’ susceptibility to TB.


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更新日期/Last Update: 1900-01-01