第一部分

研究背景和目的：输血相关急性肺损伤（transfusion-related acute lung injury，TRALI）是输血导致的严重肺部并发症，已成为输血导致死亡的主要原因之一，然而其具体的发病机制尚未完全阐明。可溶性CD40配体（soluble CD40 ligand，sCD40L）是血制品在存储过程中衍生的炎性介质并且参与多种输血不良反应的发生。本研究拟建立临床相关的TRALI大鼠模型并在该模型中探究sCD40L在TRALI中的作用。

方法：研究选用Lewis大鼠，制备大鼠悬浮红细胞，4 ℃保存0-35 d备用，并通过红细胞形态学改变、血液学指标、生化指标及代谢指标对储存不同时间的红细胞进行质量评估。通过创伤-失血-大量输血法建立TRALI大鼠模型，以大鼠股动静脉置管作为创伤打击，采集大鼠全血的30%作为失血打击，经历1 h休克期后予大鼠补液、输血，将受血大鼠随机分为5组[Day14、Day21、Day28、Day35及生理盐水（normal saline，NS）组]，分别予各组大鼠输注保存14 d、21 d、28 d、35 d的大鼠悬浮红细胞或等量NS。通过急性肺损伤（acute lung injury，ALI）三大主要特点：肺损伤病理学改变、肺泡毛细血管屏障通透性改变及肺损伤相关炎性反应的发生验证建模是否成功。通过酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）检测库血及受血大鼠血浆中sCD40L含量评估sCD40L在TRALI中的作用。

结果：红细胞在存储过程中发生了一系列形态学及生理学改变，随存储时间的延长，红细胞的存储损伤逐渐加重。输注保存35 d悬浮红细胞的大鼠肺组织可见大量中性粒细胞浸润且肺泡间隔明显增厚；Day35组大鼠支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中总蛋白含量、伊文思蓝染液（Evans blue dye，EBD）漏出量、肺损伤相关炎性因子表达量显著增加（P<0.05）。库血中sCD40L含量随存储时间的延长而逐渐增加（P＜0.05），受血大鼠血浆中sCD40L含量仅轻度增加且各组间无显著性差异（P>0.05）。

结论：通过创伤-失血-大量输血法成功建立TRALI大鼠模型；sCD40L在TRALI的发生中可能具有一定作用。

第二部分

研究背景和目的：输血相关急性肺损伤（transfusion-related acute lung injury, TRALI）是输血导致的严重肺部并发症，已成为输血导致死亡的主要原因之一，但其病理生理学机制尚未完全阐明，目前也尚无有效地特异性防治措施。肺泡毛细血管屏障的破坏是TRALI发生的共同途径。血管内皮生长因子A（vascular endothelial growth factor A，VEGFA）是调节血管通透性的关键效应分子，可通过其主要信号传导受体（VEGF receptor 2，VEGFR2）发挥生物学效应。本研究拟建立TRALI小鼠模型并在该模型中探究VEGFA在TRALI中的作用及可能存在的相关机制。

方法：研究选用BALB/c小鼠，将小鼠随机分为4组（Naïve组、LPS对照组、同型对照组、TRALI组），通过腹腔注射脂多糖（lipopolysaccharide，LPS）联合尾静脉注射MHC I类抗体（34-1-2S）建立TRALI小鼠模型。通过急性肺损伤（acute lung injury，ALI）三大主要特点：肺损伤病理学改变、肺泡毛细血管屏障通透性改变及肺损伤相关炎性反应的发生验证建模是否成功。通过Western blot法检测小鼠肺组织VEGFA及VEGFR2信号通路蛋白表达量评估VEGFA在TRALI中的作用及VEGFR2信号通路活化情况。

结果：与其余各组相比，TRALI组（LPS+34-1-2S）小鼠存在明显的呼吸困难，且活动量明显减少，小鼠死亡率显著增高（P＜0.01），肺体积明显增大，肺组织表面可见明显淤血改变，镜下可见肺泡间隔明显增厚，肺泡腔内有大量均匀红染物质（水肿液），肺组织存在大量中性粒细胞浸润，肺组织湿干重比、支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中总蛋白含量、肺损伤相关炎性因子表达量显著增高（P<0.05）。TRALI组小鼠肺组织VEGFA表达量显著高于其余各组（P＜0.05），VEGFR2-SRC-AKT信号通路存在明显活化；VEGFR2-MAPKs信号通路存在部分活化。

结论：联合LPS和MHC I类抗体成功建立TRALI小鼠模型；VEGFA在TRALI的发生中可能具有一定促进作用，该作用可能是通过调节VEGFR2信号传导通路实现的。

Part I

Background and Objectives Transfusion-related acute lung injury (TRALI) is a serious pulmonary complication of transfusion and has been one of the leading causes of transfusion-associated fatalities. However, the pathogenesis of TRALI is still unclear. Soluble CD40 ligand (sCD40L) is a proinflammatory cytokine that derived from blood component during storage and is involved in a variety of adverse transfusion reactions. The objective of this study was to establish a clinically relevant TRALI rat model and to evaluate the role of sCD40L in TRALI.

Methods Lewis rats were used. Rats’ red blood cell (RBC) suspensions were prepared and stored at 4 ℃ up to 35-day. The quality of RBC were evaluated by morphological changes, hematological indexes, biochemical indexes and metabolic indexes. A trauma-hemorrhage-transfusion strategy was applied to build the TRALI rat model. Rats were implanted with femoral artery and venous catheters and then subjected to 30% controlled arterial hemorrhage. After 60-minute shock period, rats were resuscitated with crystalloid and RBC suspensions. The blood recipients were randomly divided into 5 groups [Day14, Day21, Day28, Day35, and normal saline (NS) groups], and received RBC suspensions stored for 14 d, 21 d, 28 d, and 35 d or equal amount of NS, respectively. Lung edema was evaluated by histopathology examination, bronchoalveolar lavage fluid (BALF) protein accumulation, Evans blue dye (EBD) leakage and inflammatory cytokine concentrations. The enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of sCD40L in the RBC suspensions and recipients’ plasma to evaluate the role of sCD40L in TRALI.

Results RBCs experience a series of morphological and physiological changes during storage. The storage lesions of RBCs gradually increased over time. Obvious histological evidence of lung injury of rats transfused with a 35-day RBC suspension were observed. The BALF protein accumulation, EBD leakage, lung injury associated inflammatory cytokines concentration were increased significantly in the Day35 group (P<0.05). The sCD40L concentration increased significantly in the storage RBC suspensions over time (P<0.05) but was slightly elevated in rat plasma (P>0.05).

Conclusions These findings indicated successful establishment of a TRALI rat model with trauma-hemorrhage-transfusion, in which sCD40L may play a minor role in the development of TRALI.

Part II

Background and Objectives Transfusion-related acute lung injury (TRALI) is a serious pulmonary complication of transfusion and has been one of the leading causes of transfusion-associated fatalities. However, the pathogenesis of TRALI is still unclear, and there is currently no effective and specific control measures. The destruction of the alveolar capillary barrier is a common way of TRALI. Vascular endothelial growth factor A (VEGFA) is a key effector molecule that regulates vascular permeability, and can exert its biological effects through its main signaling receptors VEGF receptor 2 (VEGFR2). The objective of this study was to establish a TRALI mouse model and evaluate the role of VEGFA in TRALI and possible related mechanisms.

Methods BALB/c mice were used and randomly divided into 4 groups (Naïve group, LPS control group, isotype control group, TRALI group). Intraperitoneal injection of lipopolysaccharide (LPS) combined with tail vein injection of MHC class I antibody (34-1-2S) was applied to establish TRALI mouse model. Lung edema was evaluated by histopathology examination, lung wet/dry ratio, bronchoalveolar lavage fluid (BALF) protein accumulation and inflammatory cytokine concentrations. The expression of VEGFA and VEGFR2 signaling pathway proteins in mouse lung tissue was measured by western blot to evaluate the role of VEGFA in TRALI and the activation of VEGFR2 signaling pathway.

Results Compared with the other groups, mice in the TRALI group (LPS+34-1-2S) had significant dyspnea and decreased activity. The mortality rate of the mice in TRALI group was significantly increased (P<0.01). The lung volume of the mice in TRALI group was significantly increased, there was obvious congestion changes on the lung surface. Obvious thickening of the alveolar interval, a large amount of uniform red-stained substance (edema fluid) in the alveolar cavity, a large amount of neutrophil infiltration were observed under the microscope. The lung wet/dry ratio, BALF protein accumulation and inflammatory cytokine concentrations in TRALI group were significantly increased (P<0.05). The expression of VEGFA in lung tissue of mice in TRALI group was significantly higher than that in the other groups (P<0.05). VEGFR2-SRC-AKT signaling pathway was significantly activated and VEGFR2-MAPKs signaling pathway was partially activated.

Conclusions A TRALI mouse model was successfully established through the combination of LPS and MHC class I antibodies. VEGFA may play a role in the development of TRALI, which may be achieved by regulating the VEGFR2 signaling pathway.