目的 探讨张力反应性增强因子结合蛋白(tonicity-responsive enhancer binding protein, TonEBP)基因对巨噬细胞活性、迁移、增殖及吞噬功能的影响。方法 利用TonEBP-shRNA慢病毒感染Raw 264.7细胞,筛选稳定细胞株,Real time-PCR、Western blot评估干扰效率,选取干扰效率最高的慢病毒感染组及空载慢病毒感染组用于后续实验。各组细胞同步培养,孵育24 h后CCK-8法测定细胞活性,transwell实验观察细胞迁移能力,流式细胞术检测细胞周期及吞噬功能。结果 成功建立并筛选出干扰TonEBP稳定细胞株,与正常细胞组相比,mRNA和蛋白的干扰效率均高于70％,差异具有统计学意义(P＜0.05);下调TonEBP表达可使细胞活性明显下降[24 h (0.39±0.02 vs 0.46±0.01),48 h(0.57±0.02 vs 0.63±0.02),72 h(0.71±0.02 vs 1.01±0.11),96 h(0.93±0.06 vs 1.42± 0.04),120 h(1.26±0.06 vs 1.56±0.09) ](P＜0.05),同时显著降低细胞迁移(88±7.00 vs 356±35.23)、增殖[(38.73±2.57)％ vs (50.20±2.29)％]及吞噬[(2.26±0.10)％ vs (5.63±0.42)％]能力,差异均具有统计学意义(P＜0.05)。结论 干扰TonEBP的表达可有效抑制巨噬细胞活性,降低其迁移、增殖及吞噬能力,为继续探究与巨噬细胞相关的疾病提供实验依据。
Abstract
Objective To investigate the effect of TonEBP gene on macrophage activity, migration, proliferation and phagocytosis. Methods The Raw264.7 cells transfected with TonEBP -shRNA lentivirus were screened by purinomycin to establish stable cell lines. The interference efficiency was evaluated by real-time PCR and Western blot. The lentivirus infection group with the highest interference efficiency and the empty lentivirus infection group were selected for subsequent experiments. Cells in each group were simultaneously cultured and incubated for 24 h.The cell activity was measured by CCK-8 assay, and the migration ability was observed by transwell migaration assay. Finally, the cell cycle and phagocytosis ability were detected by flow cytometry. Results The TonEBP stable cell line was successfully established and screened out. Compared with the wild type cell group, the interference efficiency of mRNA and protein was over 70% (P<0.05).Downregulation of TonEBP expression significantly reduced cell activity[24 h (0.39±0.02 vs 0.46±0.01),48 h (0.57±0.02 vs 0.63±0.02),72 h (0.71±0.02 vs 1.01±0.11),96 h (0.93±0.06 vs 1.42±0.04),120 h (1.26±0.06 vs 1.56±0.09) ] (P<0.05), red cell migration (88±7.00 vs 356±35.23), proliferation[ (38.73±2.57) ％ vs (50.20±2.29) ％] and phagocytosis [ (2.26±0.10) ％ vs (5.63±0.42) ％] (P<0.05). Conclusions Interference with TonEBP expression can effectively inhibit the activity of macrophages, reduce their migration, proliferation and phagocytosis ability. These results can contribute to further exploration of diseases related to macrophages.
关键词
张力反应性增强因子结合蛋白 /
Raw264.7 /
活性 /
迁移能力 /
增殖能力 /
吞噬能力
Key words
tonicity-responsive enhancer binding protein /
Raw264.7 /
activity /
migration /
proliferation /
phagocytosis
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基金
国家自然科学基金项目(81600328);天津市自然科学基金项目(16JCQNJC11800);武警后勤学院中心实验室开放基金项目(2015ZXKF11);国家重点研发计划资助(2017YFC1307600,2017YFC1307602)
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