急性呼吸窘迫综合征患者肺泡微环境对肺成纤维细胞生物学活性的影响

Effect of alveolar microenvironment on biologic activity of lung fibroblast in patients with acute respiratory distress syndrome

目的探讨不同程度急性呼吸窘迫综合征(ARDS)肺泡微环境的变化对肺成纤维细胞(LF)的影响。方法以2014年1月至2016年12月绍兴市人民医院重症监护病房(ICU)收治的58例符合ARDS诊断标准的患者,依据患者氧合指数和柏林定义标准将患者分为轻度组(26例)、中度组(15例)、重度组(17例),另纳入10例行机械通气但无肺部疾病患者作为无肺损伤组。所有入选患者均进行支气管肺泡灌洗,留取支气管肺泡灌洗液(BALF)标本,酶联免疫吸附法(ELISA)检测其中白细胞介素1β(IL-1β)、角质细胞生长因子(KGF)和肝细胞生长因子(HGF)水平,并进行蛋白定量及细胞计数。同时,体外培养肺成纤维细胞系MRC-5细胞,与各组患者BALF共培养,采用Transwell法检测细胞迁移能力,采用Western-blotting法检测各组细胞CollagenⅠ和血小板源性生长因子α受体(PDGF-Rα)表达。结果与无肺损伤组比较,ARDS轻度组、中度组及重度组患者的IL-1β[12(8,21)、776(449,943)、802(691,1 004)、886(548,1 130)ng/L]、KGF[2.0(1.0,2.2)、19.0(15.8,24.5)、41.4(36.2,57.3)、64.7(49.2,82.1)ng/L]、HGF[90(75,106)、514(373,785)、867(674,1 248)、940(660,1 344)ng/L]、蛋白含量[(0.09±0.03)、(0.29±0.10)、(0.45±0.09)、(0.68±0.16)g/L]及细胞总数[(2.4±0.6)、(16.5±2.1)、(17.8±2.0)、(18.2±2.0)×109个/m L]均明显升高(Z=26.182、55.871、36.354,F=72.860、177.291,P均<0.05),且与重度组患者比较,轻度组及中度组患者的KGF水平及蛋白含量显著降低(P均<0.05),轻度组仅HGF水平及总细胞数明显降低(P均<0.05),而ADRS各组间IL-1β比较,差异均无统计学意义(P均>0.05)。同时,与无肺损伤组比较,ARDS各组间趋化指数均明显升高(F=12.291,P=0.003),且重度ARDS组患者明显高于轻度及中度组患者(P均<0.05)。四组间CollagenⅠ蛋白及PDGFRα蛋白比较,差异均有统计学意义(F=358.943,P=0.001;F=4.574,P=0.002)。ARDS各组CollagenⅠ蛋白明显高于无肺损伤组,且中度、重度组明显高于轻度组(P均<0.05)。而PDGF-Rα蛋白在无肺损伤组和轻度组几乎无表达,但在中度、重度组均有明显表达(P均<0.05)。结论随着ARDS程度的加重,BALF中KGF、HGF明显上升,BALF可通过诱导LF表达PDGF-Rα和CollagenⅠ,促进LF的迁移与分化。

Objective To evaluate the effect of changes in alveolar microenvironment on lung fibroblast（LF） in patients with acute respiratory distress syndrome（ARDS）. Methods Fifty-eight ARDS patients admitted to the intensive care unit（ICU） of Shaoxing People＇s Hospital from January 2014 to December 2016 were recruited. The patients were divided into the mild group（n = 26）, moderate group（n = 15） and severe group（n = 17） according to patients＇ oxygenation index and Berlin definition criteria. Another 10 patients with mechanical ventilation but no pulmonary diseases were enrolled as the control group. All patients were received bronchoalveolar lavage and the bronchial alveolar lavage fluid（BALF） was collected. The levels of interleukin-1β（IL-1β）, keratinocyte growth factor（KGF） and hepatocyte growth factor（HGF） in the BALF were measured by enzyme linked immunosorbant assay（ELISA）, and the protein content and cell counts in the BALF were also recorded. Lung fibroblasts（MRC-5） were cultured in vitro and co-cultured with BALF in each group. Cell migration assays were performed by the Transwell system. The expressions of Collagen Ⅰ and platelet-derived growth factor-receptor α（PDGF-Rα） were determined by Western-blotting. Results Compared with the control group, the levels of IL-1β [12（8, 21）, 776（449, 943）, 802（691, 1 004）, 886（548,1 130） ng/L], KGF [2.0（1.0, 2.2）, 19.0（15.8, 24.5）, 41.4（36.2, 57.3）, 64.7（49.2, 82.1） ng/L],HGF [90（75, 106）, 514（373, 785）, 867（674, 1 248）, 940（660, 1 344） ng/L], the protein content [（0.09 ± 0.03）,（0.29 ± 0.10）,（0.45 ± 0.09）,（0.68 ± 0.16） g/L] and cell counts [（2.4 ± 0.6）,（16.5 ± 2.1）,（17.8 ± 2.0）,（18.2 ± 2.0） × 109/m L] in mild, moderate and severe groups increased significantly（Z = 26.182, 55.871, 36.354; F = 72.860, 177.291; all P〈0.05）. The KGF level and protein content in mild and moderate groups were much lower, and the HGF and cell counts in the mild group were much lower as compared with severe group（all P〈0.05）.However, there were no significant differences in the IL-1β level among each ARDS subgroups（all P〈0.05）. Compared with the control group, the cell migration assays in each ARDS subgroups were much higher（F = 12.291, P = 0.003）, and they were highest in severe group（all P〈0.05）. The expressions of CollagenⅠand PDGF-Rα showed significant differneces among four groups（F = 358.943, P = 0.001; F = 4.574, P = 0.002）. The expression of Collagen Ⅰ in each ARDS subgroups was much higher than that in the control group, and it was much higher in moderate and severe groups than in mild group（all P〈0.05）. The expression of PDGF-Rαincreased obviously in moderate and severe groups, but it was almost absent in control and mild groups（all P〈0.05）. Conclusion With the aggravation of ARDS, the levels of KGF and HGF in BALF increased obviously, and the BALF in ARDS patients could induce LF migration and differentition through elevated CollagenⅠ and PDGF-Rα expressions.