Lou Xiangyu, Chen Yulong, Liu Xuening, Wu Yaosong, Li Chenxu, Shang Yiwan, Gao Xiaoling, Cui Shanshan
Henan University of Traditional Chinese Medicine (TCM), Henan Key Laboratory of TCM Prescription and Syndrome Signaling, Zhengzhou 450046, Henan, China. Corresponding author: Cui Shanshan, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2021 Jan;33(1):38-42. doi: 10.3760/cma.j.cn121430-20200820-00587.
To screen the time points of high survival rate and efferocytosis dysfunction of rat alveolar macrophages stimulated by cigarette smoke extract (CSE), establish an in vitro model of alveolar macrophage efferocytosis function, and study chronic respiratory diseases with chronic inflammatory reaction as the main pathological changes.
(1) Time point screening experiment: rat alveolar macrophages (NR8383 cells) were cultured in vitro, and the cells in logarithmic growth phase were divided into blank control group (100 μL complete medium) and 5% CSE group (90 μL complete medium + 10 μL 100% CSE). Alma blue method was used to detect the effect of 5% CSE on the activity of NR8383 cells at 6, 12, 24 and 48 hours. (2) Apoptosis induction experiment: rat type II alveolar epithelial cells (RLE-6TN cells) were cultured in vitro as phagocytic target cells of NR8383 cells, and the cells in logarithmic growth phase were divided into blank control group and 10, 30 and 60 minutes groups after ultraviolet exposure (apoptosis was induced by 30 000 μJ/cm ultraviolet irradiation for 15 minutes). Flow cytometry was used to detect the apoptosis rate of RLE-6TN cells cultured for 10, 30 and 60 minutes after ultraviolet exposure. (3) Cell efferocytosis experiment: NR8383 cells in logarithmic phase were divided into blank control group and 5% CSE group. Two hours before NR8383 cells were stimulated by CSE for 6, 12 and 24 hours, RLE-6TN cells were exposed to ultraviolet to induce apoptosis, and the RLE-6TN cell suspension was added to NR8383 cells (the ratio of RLE-6TN cells to NR8383 cells was 5:1). Flow cytometry was used to detect the efferocytosis rate of NR8383 cells to RLE-6TN cells at different time points treated with 5% CSE.
(1) Compared with the blank control group, the activity of NR8383 cells significantly decreased after treatment with 5% CSE for 48 hours [cell reduction rate: (68.5±4.1)% vs. (73.6±2.3)%, P < 0.05]. However, there were no significant differences when the activities of NR8383 cells treated with 5% CSE for 6, 12 and 24 hours were compared with the blank control group, so these three time points were selected for the subsequent establishment of alveolar macrophage cell efferocytosis dysfunction in vitro model experiment. (2) Compared with the blank control group, the apoptosis rate of RLE-6TN cells significantly increased at 10, 30 and 60 minutes after ultraviolet exposure [(66.87±8.63)%, (85.51±2.39)%, (96.13±2.74)% vs. (9.13±3.17)%, all P < 0.01] in a time-dependent manner. Considering that it taked about 50 minutes for RLE-6TN cells to be labeled with PKH26 membrane labeling probe, 10 minutes after ultraviolet exposure was selected to label RLE-6TN cells. (3) Compared with the blank control group, the efferocytosis function of NR8383 cells was significantly decreased after treatment with 5% CSE for 12 hours [cell efferocytosis rate: (33.64±1.30)% vs. (44.02±2.71)%, P < 0.01], but there was no significant effect on the efferocytosis function of NR8383 cells at 6 hours and 24 hours.
CSE can induce alveolar macrophage cell efferocytosis dysfunction. Based on the test results of the effect of 5% CSE on NR8383 cell activity and cell efferocytosis function, 12 hours with high survival rate and weak efferocytosis effect of NR8383 cells can be selected as the in vitro model condition of alveolar macrophage cell efferocytosis dysfunction.
筛选香烟烟雾提取物（CSE）刺激大鼠肺泡巨噬细胞后高存活率及胞葬作用功能障碍的时间点，建立肺泡巨噬细胞胞葬作用功能的体外模型，研究以慢性炎症反应为主要病理变化的慢性呼吸道疾病。
（1）时间点筛选实验：体外培养大鼠肺泡巨噬细胞（NR8383细胞），将对数生长期细胞分为空白对照组（100 μL完全培养基）和5% CSE组（90 μL完全培养基 + 10 μL 100% CSE）。采用阿尔玛蓝法检测5% CSE在6、12、24和48小时对NR8383细胞活性的影响。（2）凋亡诱导实验：体外培养大鼠Ⅱ型肺泡上皮细胞（RLE-6TN细胞）作为NR8383细胞的吞噬靶细胞，将对数生长期细胞分为空白对照组和紫外线照射后10、30和60分钟组（30 000 μJ/cm紫外线照射诱导凋亡15分钟）。采用流式细胞术检测紫外线照射后培养10、30和60分钟的RLE-6TN细胞凋亡率。（3）细胞胞葬作用实验：将对数期NR8383细胞分为空白对照组和5% CSE组。在NR8383细胞被CSE刺激6、12和24小时前2小时，对RLE-6TN细胞进行紫外线照射诱导凋亡，并将RLE-6TN细胞悬液加入NR8383细胞（RLE-6TN细胞与NR8383细胞比例为5:1）。采用流式细胞术检测5% CSE处理不同时间点NR8383细胞对RLE-6TN细胞的胞葬作用率。
（1）与空白对照组相比，5% CSE处理48小时后NR8383细胞活性显著降低[细胞减少率：(68.5±4.1)% vs. (73.6±2.3)%，P < 0.05]。然而，5% CSE处理6、12和24小时的NR8383细胞活性与空白对照组相比无显著差异，因此选择这三个时间点进行后续体外建立肺泡巨噬细胞胞葬作用功能障碍模型实验。（2）与空白对照组相比，紫外线照射后10、30和60分钟RLE-6TN细胞凋亡率显著升高[(66.87±8.63)%、(85.51±2.39)%、(96.13±2.74)% vs. (9.13±3.17)%，均P < 0.01]，呈时间依赖性。考虑到RLE-6TN细胞用PKH26膜标记探针标记约需50分钟，选择紫外线照射后10分钟标记RLE-6TN细胞。（3）与空白对照组相比，5% CSE处理12小时后NR8383细胞的胞葬作用功能显著降低[细胞胞葬作用率：(33.64±1.30)% vs. (44.02±2.71)%，P < 0.01]，但在6小时和24小时对NR8383细胞的胞葬作用功能无显著影响。
CSE可诱导肺泡巨噬细胞胞葬作用功能障碍。基于5% CSE对NR8383细胞活性和细胞胞葬作用功能的检测结果，可选择NR8383细胞存活率高且胞葬作用效应弱的12小时作为肺泡巨噬细胞胞葬作用功能障碍的体外模型条件。
