辐射后A549细胞上清外泌体TMEM88蛋白表达与细胞周期的相关性

Relationship between cell cycle and exosome TMEM88 protein in supernatant of lung adenocarcinoma A549 cells after irradiation

目的探讨辐射对肺腺癌A549细胞细胞周期的影响,观察其细胞上清外泌体TMEM88蛋白(transmembrane protein 88)的表达情况。方法取对数生长期肺腺癌A549细胞,按0、0.5、1、1.5、2 Gy剂量(剂量率1.75 Gy/min,300 Mu/min)照射细胞,培养6和12 h后,流式细胞术检测不同辐射剂量对细胞周期的影响;收集细胞上清,超速离心提取外泌体蛋白,Western blot法分析外泌体TMEM88蛋白的表达情况。结果 G2期细胞6 h检测结果显示,与0 Gy组相比,各剂量组细胞比例显著增加(P<0.001),且随照射剂量增加,细胞比例增加越明显,除0.5 Gy组与1 Gy组外,其余各组间差异均有统计学意义(P<0.001);12 h检测结果显示,G2期细胞比例随照射剂量增加而显著增加,各组间差异均有统计学意义(P<0.05)。S期细胞6 h检测结果显示,与0 Gy组相比,0.5、1.0、1.5 Gy组S期细胞比例显著增加(P<0.05);12 h检测结果显示,与0、0.5 Gy组相比,1.0、1.5 Gy组S期细胞比例显著增加(P<0.05)。随照射剂量的增加,外泌体TMEM88蛋白的表达逐渐降低,6 h检测结果显示,0 Gy组与1.5、2.0 Gy组以及0.5 Gy组与2.0 Gy组相比,差异均有统计学意义(P<0.05));12 h检测结果显示,0.5、1.0、1.5、2.0 Gy组与0 Gy组相比,差异均有统计学意义(P<0.001)。结论辐射能够诱导肺腺癌A549细胞细胞周期阻滞,并能抑制细胞上清外泌体TMEM88蛋白的表达。

Objective To investigate the effect of radiation on the cell cycle of lung adenocarcinoma A549 cells and observe the expression of exosome TMEM88 protein in supernatant. Methods Lung adenocarcinoma A549 cells in logarithmic growth phase were radiated at dosages of 0, 0. 5, 1, 1. 5 and 2 Gy（1. 75 Gy/min, 300 Mu/min）, then cultured for 6 and 12 h. The effect of radiation dosage on cell cycle was measured by flow cytometry. The cell supernatant was collected, from which the exosomes were extracted by ultracentrifugation and determined for expression of TMEM88 protein by Western blot. Results The result of flow cytometry 6 h after culture showed that, compared with that radiated at a dosage of 0 Gy, the proportion of cells in G2 phase after radiation at various dosages increased significantly（P〈0. 001）, and increased with the increasing dosage, which showed significant difference in various groups except those radiated at 0. 5 and 1 Gy（P〈0. 001）. However, the result of flow cytometry 12 h after culture showed that the proportion of cells at G2 phase increased significantly with the increasing irradiation dosage, which showed significant difference in various groups（P〈0. 05）. The result of flow cytometry 6 h after culture showed that, compared with that radiated at a dosage of 0 Gy, the proportion of cells in S phase after radiation a dosages of 0. 5, 1 and 1. 5 Gy increased significantly（P〈0. 05）. However, the result of flow cytometry 12 h after culture showed that, compared with that radiated at dosages of 0 and 0. 5 Gy, the proportion of cells in S phase after radiation at dosages of 1 and 1. 5 Gy increased significantly（P〈0. 05）. The expression level of TMEM88 protein decreased with the increasing radiation dosage. Six hours after culture,the expression level in 0 Gy group showed significant difference with those in 1. 5 and 2. 0 Gy groups, while that in 0. 5 Gy group showed significant difference with that in 2. 0 Gy group（each P〈0. 05）. However, 12 h after culture, the expression levels in 0. 5, 1. 0, 1. 5 and 2. 0 Gy groups showed significant difference with that in 0 Gy group（P〈0. 001）.Conclusion Radiation induced the cell cycle arrest of A549 cells and inhibited the expression of exosome TMEM88 protein in supernatant.