肺泡巨噬细胞胞葬功能障碍体外模型建立

Establishment of an in vitro model of alveolar macrophage cell efferocytosis dysfunction

目的通过筛选香烟烟雾提取物(CSE)刺激下大鼠肺泡巨噬细胞存活率高、胞葬功能弱的时间点,建立肺泡巨噬细胞胞葬功能障碍体外模型,用于研究以慢性炎症反应为主要病理变化的慢性呼吸系统疾病。方法①时间点筛选实验:体外培养大鼠肺泡巨噬细胞株NR8383,取对数生长期细胞分为空白对照组(100μL完全培养基)和5%CSE组(90μL完全培养基+10μL 100%CSE);采用阿尔玛蓝法检测5%CSE作用6、12、24、48 h对NR8383细胞活性的影响。②诱导凋亡实验:体外培养大鼠Ⅱ型肺泡上皮细胞株RLE-6TN作为NR8383细胞的吞噬靶细胞,取对数生长期细胞分为空白对照组及紫外线暴露后10、30、60 min组(以30000μJ/cm2紫外线强度照射15 min诱导凋亡);采用流式细胞仪检测RLE-6TN细胞在紫外线暴露结束后10、30、60 min的凋亡率。③胞葬实验:另取对数生长期NR8383细胞分为空白对照组和5%CSE组。于CSE刺激NR8383细胞达到6、12、24 h前2 h,将RLE-6TN细胞进行紫外线暴露诱导凋亡;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)。采用流式细胞仪检测5%CSE作用不同时间点NR8383细胞对RLE-6TN细胞的胞葬率。结果①与空白对照组相比,5%CSE作用48 h后NR8383细胞活性明显降低〔细胞还原率:(68.5±4.1)%比(73.6±2.3)%,P<0.05〕;而5%CSE作用6、12、24 h对NR8383细胞活性的影响与空白对照组比较差异均无统计学意义,故选择这3个时间点用于后续建立肺泡巨噬细胞胞葬功能障碍体外模型实验。②与空白对照组相比,紫外线暴露后10、30、60 min时RLE-6TN细胞凋亡率均显著升高〔(66.87±8.63)%、(85.51±2.39)%、(96.13±2.74)%比(9.13±3.17)%,均P<0.01〕,并且呈一定时间依赖性。考虑在胞葬实验中PKH26膜标记探针标记RLE-6TN细胞大约需50 min,因此,选择在紫外线暴露后10 min进行RLE-6TN细胞标记。③与空白对照组相比,5%CSE作用12 h时NR8383细胞的胞葬功能明显降低〔细胞胞葬率:(33.64±1.30)%比(44.02±2.71)%,P<0.01〕,而作用6 h、24 h时对NR8383细胞的胞葬功能无明显影响。结论CSE可诱导肺泡巨噬细胞胞葬功能障碍,基于5%CSE对NR8383细胞活性、胞葬功能影响的检测结果,可选择NR8383细胞存活率高、胞葬作用弱的12 h作为肺泡巨噬细胞胞葬功能障碍体外模型条件。

Objective 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.Methods①Time 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(100μL complete medium)and 5%CSE group(90μL complete medium+10μL 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.②Apoptosis induction experiment:rat typeⅡalveolar 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 30000μJ/cm2 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.③Cell 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.Results①Compared 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.②Compared 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.③Compared 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.Conclusions 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.