输血相关急性肺损伤对大鼠血浆和肺组织血管生成素-2表达的影响

Expression of angiopoietin-2 in plasma and lung tissue of rats with transfusion-related acute lung injury

目的探讨输血相关急性肺损伤(TRALI)时血浆和肺组织中血管生成素-2(Ang-2)的表达及其意义。方法选择60只SD大鼠,按随机数字表法分为生理盐水(NS)对照组、脂多糖(LPS)对照组、TRALI模型组、急性肺损伤(ALI)对照组,每组15只。采用"LPS-血浆二次打击"法复制TRALI大鼠模型。NS对照组腹腔注射NS 2 mL,移除大鼠血液1 mL后输注等体积NS。LPS对照组大鼠腹腔注射2 mg/kg LPS(1 min内注完)1 mL 2 h后静脉输注等体积NS。TRALI模型组腹腔注射2 mg/kg LPS 1 mL,2 h后静脉输注血浆1 mL。ALI对照组静脉输注5 mg/kg LPS 1 mL诱导ALI。制模完成后处死大鼠取肺组织观察病理学改变并进行肺损伤评分;取支气管肺泡灌洗液(BALF)检测细胞总数、中性粒细胞比例(NEUT);用蛋白质免疫印迹试验(Western Bolt)检测血浆中Ang-2蛋白表达;免疫组化法观察肺组织中Ang-2阳性表达;实时荧光定量反转录-聚合酶链反应(RT-PCR)检测大鼠肺组织中Ang-2 mRNA表达水平。结果光镜下可见,NS对照组大鼠肺组织结构清楚,肺泡壁结构完整,肺泡内未见炎性细胞渗出及肺泡萎陷,肺泡间隔均一无增宽;LPS对照组肺泡间隔轻度增宽、有少量炎性细胞浸润;TRALI模型组肺泡间隔增宽、中等量出血并有较多炎性细胞浸润;ALI对照组肺内大量炎性细胞浸润,间质水肿加重,伴出血、微血栓及灶性肺不张。ALI对照组、TRALI模型组血浆Ang-2蛋白和肺组织Ang-2 mRNA表达均显著高于NS对照组、LPS对照组[Ang-2蛋白(灰度值):0.58±0.09、0.43±0.07比0.12±0.03、0.20±0.05,Ang-2 mRNA(2^(-ΔΔCt)):0.39±0.10、0.29±0.09比0.10±0.05、0.16±0.04,P均<0.01];ALI对照组、TRALI模型组BALF细胞总数(×10~6/L:361±78、310±76比101±63、165±65)、NEUT(0.396±0.125、0.335±0.115比0.098±0.035、0.114±0.041)均明显高于NS对照组和LPS对照组(P均<0.05);ALI对照组、TRALI模型组病理学评分、肺湿重/体重比值和动脉血氧分压(PaO_2)均显著高于NS对照组和LPS对照组(P均<0.05)。ALI对照组与TRALI模型组各项指标比较差异无统计学意义(P均>0.05)。结论TRALI大鼠肺组织和血浆中Ang-2的表达升高,提示Ang-2可能参与了TRALI病理生理过程。

Objective To study the expression of angiopoietin-2 （Ang-2） in plasma and lung tissue of rats with transfusion-related acute lung injury （TRALI） and its significance. Methods Sixty Sprague-Dawley （SD） rats were randomly divided into normal saline （NS） control group, lipopolysaccharide （LPS） control group, TRALI model group, and acute lung injury （ALI） control group, with 15 rats in each group. The rat model of TRALI was reproduced by ＂LPS-plasma two-hit＂ method. The rats in NS control group were intraperitoneally injected 2 mL NS, the same volume of NS was intravenously injected after 1 mL blood collection. The rats in LPS control group were intraperitoneally injected 2 mg/kg LPS 1 mL within 1 minute, and the same volume of NS was intravenously injected 2 hours later. The rats in TRALI modelgroup were challenged by intraperitoneal injection of 2 mg/kg LPS 1 mL at 2 hours before the transfusion of 1 mL plasma. The rats in ALI control group were challenged by intraperitoneal injection of LPS 5 mg/kg to reproduce the model of ALI. The lung tissues were harvested to observe the pathological changes, and lung injury score was evaluated. The total cells and neutrophils （NEUT） in bronchoalveolar lavage fluid （BALF） were determined. Western Blot was used to detect Ang-2 protein expression in plasma, and immunohistochemical method was used to detect Ang-2 positive expression in lung tissue. The real-time fluorescent reverse transcription-quantitative polymerase chain （RT-PCR） was used to detect Ang-2 mRNA expression in the lung tissue. Results Under the light microscope, rat lung tissue structure in the NS control group was clear, the alveolar wall was intact, no alveolar exudate, alveolar collapse or inflammatory cells in alveolar septum was found, and alveolar septum did not increase. The alveolar septum was widened slightly in LPS control group, a small amount of inflammatory cell infiltration was found. The alveolar septum, moderate bleeding and more infiltration of inflammatory cells were found in TRALI model group. Lung inflammatory infiltration, interstitial edema, hemorrhage, micro-thrombosis and focal atelectasis were found in the ALI control group. Compared with NS control group and LPS control group, Ang-2 protein expression in plasma and Ang-2 mRNA expression in the lung tissue were significantly increased in ALI control group and TRALI model group [Ang-2 protein （gray ralue）： 0.58 ± 0.09, 0.43±0.07 vs. 0.12 ± 0.03, 0.20±0.05, Ang-2 mRNA （2-△△Ct）： 0.39 ± 0.10, 0.29 ± 0.09 vs. 0. 10± 0.05, 0.16 ± 0.04, P all 〈 0.01]. The total cells [total cells （× 10^6/L： 361 ±78, 310±76 vs. 101 ±63, 165±65） and NEUT （0.396±0.125, 0.335±0.115 vs. 0.098±0.035, 0.114 ± 0.041）] in BALF of ALI control group and TRALI model group were significantly higher than those of NS control group and LPS control group （P all 〈 0.05）. The pathological score, wet lung weight/body weight ratio and arterial partial pressure of oxygen （PaO：） in ALI control group and TRALI model group were significantly higher than those of NS control group and LPS control group （P all 〈 0.05）. There were no significant differences in above parameters between ALI control group and TRALI model group （P all 〉 0.05）. Condusions Expression of Ang-2 in the lung tissue and plasma were increased in rats with TRALI. Ang-2 may be involved in the TRALI pathophysiological process.