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目的探讨膜融合相关蛋白TECPR1对羊种布鲁氏菌16M感染小鼠巨噬细胞RAW264.7(RAW264.7)引起的非典型自噬及布鲁氏菌胞内生存繁殖能力的影响。方法根据AIR核苷酸序列,分别设计2条特异性小干扰RNA(short interfering RNA,siRNA)分子,亚克隆到慢病毒pLentiLox3.7载体,并转染RAW264.7,通过QRT-PCR筛选干扰效果最优的质粒;用布鲁氏菌感染干扰AIR后的RAW264.7,采用QRT-PCR检测细胞自噬相关基因p62、LC3-Ⅰ、LC3-Ⅱ的mRNA表达量,采用Western blot检测细胞自噬相关基因p62、LC3-Ⅰ、LC3-Ⅱ蛋白表达量;同时检测16M在干扰AIR后RAW264.7中的生存繁殖能力。结果获得2条对AIR干扰效果达70%以上特异性的siRNA分子,自噬相关基因LC3-Ⅰ、LC3-Ⅱ、p62的mRNA相对表达量分别为66%、77%和63%,差异有统计学意义(F值分别为21.52,25.38,10.87,P<0.05);LC3-Ⅰ、LC3-Ⅱ、p62蛋白相对表达量分别为45.71%、34.29%和46.13%,差异有统计学意义(F值分别是753.92,332.38,768.15,P<0.01);24h后干扰AIR后的RAW264.7中布鲁氏菌数量为8.58×105CFU,对照组为6.02×106 CFU,差异有统计学意义(F值为13.45,P<0.05)。结论 AIR基因沉默可促进布鲁氏菌诱导的非典型自噬进一步成熟,布鲁氏菌胞内繁殖力显著降低,这可能是AIR结构域在自噬小体和溶酶体融合过程中发挥重要作用,该研究进一步解释了布鲁氏菌能够逃避巨噬细胞自噬-溶酶体降解途径的分子机制。
Objective To investigate the effect of TECPR1, a membrane fusion protein, on the atypical autophagy induced by RAW264.7 (RAW264.7) in mice infected with Brucella abortus 16M and the intracellular viability of Brucella. Methods According to AIR nucleotide sequences, two siRNAs were designed and subcloned into the lentiviral vector pLentiLox3.7 and transfected into RAW264.7 cells. The interference effect was screened by QRT-PCR The expression of autophagy-related genes p62, LC3-I and LC3-II were detected by QRT-PCR. The expression of autophagy was detected by Western blot Related genes p62, LC3-Ⅰ, LC3-Ⅱ protein expression; simultaneous detection of 16M interference AIR after RAW264.7 survival and reproduction ability. Results Two siRNAs with more than 70% specificity for AIR interference were obtained. The mRNA relative expression of LC3-Ⅰ, LC3-Ⅱ and p62 were 66%, 77% and 63%, respectively (F = 21.52,25.38,10.87, P <0.05). The relative expression levels of LC3-Ⅰ, LC3-Ⅱ and p62 proteins were 45.71%, 34.29% and 46.13%, respectively, with significant difference Respectively, 753.92,332.38,768.15, P <0.01). The number of brucella in RAW264.7 after interference with AIR for 24h was 8.58 × 105CFU, while the control group was 6.02 × 106 CFU, the difference was statistically significant 13.45, P <0.05). Conclusion AIR gene silencing can promote the further maturation of atypical autophagy induced by Brucella, and significantly reduce the intracellular fertility of Brucella. This may be the reason that AIR domain plays an important role in the process of autophagy and lysosome fusion This study further elucidates the molecular mechanism by which Brucella escapes autophagy-lysosomal degradation of macrophages.