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目的探讨地塞米松对海水浸泡的人肺泡上皮细胞(A549)钠通道的影响。方法将常规培养的A549细胞分为海水处理组(SG)、阴性对照组(NG)和地塞米松处理组(DG)。SG组经灭菌配方海水常规孵育,DG组在经海水处理后,加入地塞米松(终浓度1μmol/L)处理,NG组常规培养至实验终点。配方海水渗透压1300mmol/L,pH8.2,相对密度1.05。采用MTT法检测细胞存活率,流式细胞仪检测细胞的凋亡和坏死情况,Western blotting法检测钠钾泵α1亚单位(NKA-α1)、钠通道α亚单位(α-ENaC)的表达,水解比色法检测NKA活性。结果流式细胞术检测结果显示,海水孵育30min后A549细胞的存活率为90.60%±2.11%,地塞米松(1μmol/L孵育6h)处理对海水孵育后细胞的存活率无影响。NG组的NKA-α1和α-ENaC蛋白表达(0.113±0.041、0.396±0.124)显著高于SG组(0.038±0.009、0.093±0.038;P<0.01)或DG组(0.067±0.015、0.165±0.078;P<0.01),但DG组显著高于SG组(P<0.05)。与NG组[31.298±5.779μmol/(mg·h)]比较,SG组NKA酶活性[14.211±3.885μmol/(mg·h)]明显降低(P<0.05);DG组的NKA酶活性[18.641±1.921μmol/(mg·)]较SG组有升高(P<0.05)。结论在不影响人肺泡上皮细胞存活状况的情况下,地塞米松可以抑制海水孵育导致的α-ENaC、NKA-α1含量及NKA活性的下降。
Objective To investigate the effect of dexamethasone on sodium channel in human alveolar epithelial cells (A549) soaked in seawater. Methods A549 cells were divided into seawater treatment group (SG), negative control group (NG) and dexamethasone treatment group (DG). The SG group was routinely incubated with sterile formula seawater. DG group was treated with seawater and then treated with dexamethasone (final concentration of 1μmol / L). NG group was routinely cultured to the end of the experiment. Recipe seawater osmotic pressure 1300mmol / L, pH8.2, the relative density of 1.05. Cell viability was detected by MTT assay. Apoptosis and necrosis of cells were detected by flow cytometry. Expressions of NKA-α1 and α-ENaC were detected by Western blotting, Hydrolysis colorimetric assay for NKA activity. Results The results of flow cytometry showed that the survival rate of A549 cells after seawater incubation for 30min was 90.60% ± 2.11%, while dexamethasone (1μmol / L for 6h) had no effect on the survival rate of cells after seawater incubation. The expression of NKA-α1 and α-ENaC in NG group was significantly higher than that in SG group (0.038 ± 0.009,0.093 ± 0.038; P <0.01) or DG group (0.067 ± 0.015,0.165 ± 0.078) ; P <0.01), but DG group was significantly higher than SG group (P <0.05). Compared with NG group [31.298 ± 5.779μmol / (mg · h)], NKA activity in SG group was significantly lower than that in NG group [14.211 ± 3.885μmol / (mg · h)] (P <0.05) ± 1.921μmol / (mg ·)] than the SG group (P <0.05). Conclusion Dexamethasone can inhibit the decline of α-ENaC, NKA-α1 and NKA activity induced by seawater incubation without affecting the survival of human alveolar epithelial cells.