建立多发骨折-脂多糖两次打击急性肺损伤动物模型及其相关研究

Establishing two-hit acute lung injury animal model induced by multiple fractures and lipopolysaccharide as well as its relative study

目的:建立多发骨折-脂多糖两次打击急性肺损伤小鼠动物模型并观察其组织病理学、血气各项指标的变化,进而对多发骨折并发感染后诱发急性肺损伤/急性呼吸窘迫综合征的病理生理机制进行初步探讨。方法:实验于2005-01/09在解放军第三军医大学大坪医院野战外科研究所创伤中心实验室完成。选择昆明种小鼠62只随机分为5组(空白对照组2只、脂多糖5mg组15只、脂多糖16mg组15只、双侧股骨骨折组15只、双侧股骨骨折复合脂多糖5mg组15只)。再按3,6,12,24,48h时间点将每组分为5个时相点,每时相点3只。空白对照组:麻醉后先行腹主动脉抽血做血气分析,动脉放血处死小鼠开胸取肺脏分别做组织病理学变化分析。脂多糖5mg组和脂多糖16mg组:腹腔内注射脂多糖(5mg/kg和16mg/kg),分别于注射后3,6,12,24,48h时做上述各指标检测。双侧股骨骨折组:麻醉后钳夹法双侧股骨中段闭合骨折造模,按相同时相点做上述各指标检测。双侧股骨骨折复合脂多糖5mg组:在骨折造模后6h腹腔注射脂多糖(5mg/kg)此后于相同时相点检测上述指标。结果:纳入动物62只,均进入结果分析。①各组肺组织病理学结构变化评分的比较:空白对照组(0.00±0.00)分,脂多糖5mg组(4.60±3.36)分,脂多糖16mg组(7.60±3.36)分,双侧股骨骨折组(2.00±2.55)分,双侧股骨骨折复合脂多糖5mg组(8.40±3.91)分。各组间评分有明显差异(F=4.48,P<0.05)。其中脂多糖16mg组和双侧股骨骨折复合脂多糖5mg组与空白对照组、双侧股骨骨折组和脂多糖5mg组比较差异显著(P<0.05)。②各组动脉血气指标变化比较:脂多糖5mg组主要改变为轻度代谢性酸中度,少数合并呼吸性酸中度或低氧;脂多糖16mg组和双侧股骨骨折复合脂多糖5mg组为明显代谢性酸中度,多数合并呼吸性酸中度或低氧改变,与空白对照组、双侧股骨骨折组、脂多糖5mg组各组的组间比较表明,脂多糖16mg组和双侧股骨骨折复合脂多糖5mg组可以引起明显的急性肺损伤血气改变,并随干预因素作用时间的延长逐渐加重,这两组中24,48h时相点组小鼠分别发生氧合指数≤40kPa(急性肺损伤)或≤26.67kPa(急性呼吸窘迫综合征)并Ⅱ型呼吸衰竭。结论:以双侧股骨骨折复合脂多糖5mg腹腔注射方法可以成功稳定地复制两次打击急性肺损伤动物模型。其组织病理学、血气改变符合急性肺损伤的典型病变。提示在多发骨折后所致的急性肺损伤/急性呼吸窘迫综合征发病因素中,多发骨折创伤严重程度与感染是两个重要诱因,二者所致的Ⅱ型肺泡上皮细胞损伤可能是其发病机制中的基础和关键环节。

AIM： To establish the animal model of two-hit acute lung injury （ALI） induced by multiple fractures and lipopolysaccharide （LPS）, observe the changes of lung histopathology and arterial blood gas, so as to investigate the pathophysiology mechanism of ALl and acute respiratory distress syndrome （ARDS） induced by these two factors. METHODS： The experiment was carried out in the Laboratory of Trauma Center, Institute of Battle Surgery, Daping Hospital, Third Military Medical University of Chinese PLA from January to September 2005. Totally sixtytwo Kunming mice were selected and divided randomly into 5 groups： blank control group with 2 mice, LIPS 5 mg group with 15 mice, LPS 16 mg group with 15 mice, bilateral femoral fracture group with 15 rats and bilateral femoral fracture compound LIPS 5 mg group with 15 rats. The mice in the 5 groups were divided randomly into 5 subgroups according to the 3^rh, 6^th, 12^th, 24^th and 48^th hours of the experiment. Blank control group： after anesthetized, arterial blood was drawn from abdominal aorta to perform blood gas analysis. Then mice were sacrificed haemospasia and collected pulmonary tissue samples for histopathology analysis. LPS 5 mg group and LIPS 16 mg group： LIPS was injected intraperitoneally （5 mg/kg and 16 mg/kg）, and collected above same samples at the 3^rd, 6^th, 12^th, 24^th and 48^th hours of the experiment for same analysis, respectively. Bilateral femoral fractures group： Using hemostatic forceps induced bilateral middle femoral shaft fractures, mice were sacrificed at each phase and performed same procedures. Bilateral femoral fractures compound LIPS 5 mg group： Mice was treated firstly with the inducement of bilateral femoral fractures, and then LPS was injected intraperitoneally （5 mg/kg） six hours later, and then performed same procedures. RESULTS： All 62 mice were involved in the result analysis. ① Comparison of changes of pulmonary histopathology structure in each group： It was （0.00±0.00） points in the blank control group. It was （4.60±3.36） points in the LPS 5 mg group. It was （7.60±3.36） points in the LIPS 16 mg group. It was （2.00±2.55） points in the bilateral femoral fractures group. It was （8.40±3.91） in the bilateral femoral fractures compound LIPS 5 mg group, There was significant difference of score in every group （F =4.48,P 〈 0.05）, That in the LPS 16 mg group and bilateral femoral fracture compound LIPS 5 mg group had obvious differetace with blank control group, bilateral femoral fracture group and LIX3 5 mg group （P 〈 0.05）. ②Comparison of changes of arterial blood gas in each group： That in the LPS 5 mg group mainly showed mild metabolic acidosis, and a few showed respiratory acidosis or hypoxia. That in the LPS 16 mg group and bilateral femoral fracture compound LPS 5 mg group showed obvious metabolic acidosis, mainly compound respiratory acidosis or hypoxia. Compared with blank control group, bilateral femoral fracture group, LPS 5 mg group, marked changes of arterial blood gas of ALl could be induced in the LPS 16 mg group and the bilateral femoral fracture compound LPS 5 mg group, and the affection gradually aggravated concomitance with experiment phase, and the severe phase were the 24^th and 48^th hours of the experiment and ALI/ARDS was confirmed had been induced （oxygenation index ≤ 40/26.67 kPa） as well as type Ⅱ respiratory failure. CONCLUSION： Use two-hit methods that bilateral femoral fractures compound LPS （5mg/kg） intraperitoneally injection can successfully replicate stable acute lung injury animal model. The changes of pulmonary histopathology score and arterial blood gas of this model accord with the typical changes of ALI. Degree of severity of multiple fractures and infection are two important inducements in onset of ALI/ARDS. The damage of AT-Ⅱ and secondum damage probably are conjunct developmental foundation and critical procedure.