【作者】 张曌；

【导师】 张诗海；

【作者基本信息】 华中科技大学 ， 麻醉学， 2010， 博士

【摘要】 第一部分血管内皮细胞生长因子在急性肺损伤中的表达目的探讨脂多糖(LPS)吸入导致急性肺损伤(ALI)模型中血管内皮细胞生长因子(VEGF)表达的变化情况及其与肺损伤程度的关系。方法将30只雄性BALB/c小鼠随机分为6组(n=5),分别为LPS 4h组、LPS 24h组、LPS 48h组、PBS 4h组、PBS 24h组和PBS 48h组,麻醉后行气管内插管,LPS组吸入LPS (3mg/kg), PBS组吸入等容量磷酸盐缓冲液(PBS)作为对照。在预定的时间点(4h,24h,48h)左心室穿刺,行血气分析；保留血浆检测VEGF水平；肺泡灌洗液(BALF)检测VEGF水平和蛋白定量；留取肺组织行病理学检测、免疫组化检测以及实时定量PCR检测VEGF mRNA表达水平。结果与PBS各组相比,LPS组均出现明显的肺损伤改变,其中LPS 24h组的病理变化最为明显,肺损伤评分显著增加(1.5 vs 12,P<0.001)；与PBS 24h组相比：LPS 24h组出现明显低氧血症(97.27±8.96 mmHg vs 64.24±10.53 mmHg, P<0.001)、二氧化碳潴留(37.83±5.75 mmHg vs 49.00±9.19 mmHg, P< 0.05)和酸中毒(7.42±0.07 vs 7.17±0.05, P< 0.001); BALF中蛋白定量(183.28±15.58μg/ml vs 569.17±114.68μg/ml, P<0.05)和VEGF(32.45±6.57 pg/ml vs 160.14±34.25 pg/ml, P<0.05)均明显增加,且二者呈正相关(r=0.676,P<0.001)；血浆(12.44±4.05 pg/ml vs 31.53±1.96 pg/ml, P<0.05)和肺组织中VEGF升高,肺组织中VEGF mRNA表达也升高(0.24±0.07 vs 0.55±0.10,P<0.05)。结论LPS吸入成功地建立了ALI模型,ALI模型的血浆、肺组织和BALF中VEGF水平明显升高,BALF中蛋白定量与VEGF呈正相关,提示VEGF可能通过增加呼吸膜的通透性参与ALI的病理生理过程。第二部分抑制血管内皮细胞生长因子治疗对急性肺损伤的治疗作用目的研究抗VEGF治疗对ALI病理生理变化的影响以及抗VEGF对ALI可能存在的治疗作用。方法将60只雄性BALB/c小鼠随机分为4组,分别为PBS组、sFlt组、LPS组和LPS+sFlt组,再按时间点将每组动物分为3个亚组(4h组、24h组、48h组),所有动物麻醉后行气插管,LPS组和LPS+sFlt组吸入LPS (3mg/kg), PBS组和sFlt组吸入等容量PBS,然后sFlt组和LPS+sFlt组从尾静脉给予50μg/kg的sFlt-1 (VEGF的可溶性受体,仅有与VEGF结合能力而无信号传导作用)进行抗VEGF治疗,PBS组和LPS组给予等容量PBS。在预定的时间点左心室穿刺,行血气分析；保留血浆检测VEGF水平；BALF检测VEGF水平和蛋白定量；留取肺组织行病理学检测、免疫组化检测以及实时定量PCR检测VEGF mRNA表达水平。结果PBS组和sFlt组均无肺损伤改变,与LPS各亚组相比,LPS+sFlt各亚组的血气和病理指标均有所改善,与LPS 24h组相比,LPS+sFlt 24h组的低氧血症(64.24±10.53 mmHg vs 84.31±7.23 mmHg,P<0.001)和酸中毒(7.17±0.05 vs 7.29±0.07,P<0.05)明显减轻,且无二氧化碳潴留(49.00±9.19 mmHg vs 36.51±4.83 mmHg, P<0.05);肺损伤评分(12 vs 6,P<0.05)和BALF蛋白定量(569.17±114.68μg/ml vs 264.82±76.92μg/ml,P<0.05)均明显降低；与LPS组相比,LPS+sFlt组血浆(31.53±1.96 pg/ml vs 39.69±8.98 pg/ml, P>0.05)、BALF (160.14±34.25 pg/ml vs 127.52±17.83 pg/ml, P>0.05)和肺组织中的VEGF及VEGF mRNA (0.55±0.10 vs 0.66±0.10, P>0.05)的表达均无明显变化。结论sFlt-1能够有效地改善ALI的症状,其作用机制可能是通过竞争性抑制VEGFR-1与VEGF的结合,从而抑制VEGF的作用,维持了呼吸膜的形态和功能,改善ALI模型的血气分析和病理评分,减少血浆蛋白的渗漏。第三部分抑制血管内皮细胞生长因子对肺损伤保护机制的研究目的探讨VEGF在LPS吸入导致的ALI模型中的作用机制及sFlt对ALI的保护机制。方法将60只雄性BALB/c小鼠随机分为4组,分别为PBS组、sFlt组、LPS组和LPS+sFlt组,再按时间点将每组动物分为3个亚组(4h组、24h组、48h组),所有动物麻醉后插入气管导管,LPS组和LPS+sFlt组吸入LPS (3mg/kg), PBS组和sFlt组吸入等量PBS, sFlt组和LPS+sFlt组从尾静脉给予50μg/kg的sFlt-1, PBS组和LPS组给予等量PBS。在预定的时间点放血处死动物,留取肺组织进行超微病理学检测并计算凋亡指数,实时定量PCR检测凋亡蛋白Bcl2、BAX mRNA的表达水平。结果LPS各组肺泡上皮细胞紧密连接松解,肺泡上皮通透性增加,呼吸膜肿胀增厚,sFlt-1明显改善ALI肺组织中肺泡上皮细胞的紧密连接松解,减少BALF蛋白渗漏,降低呼吸膜厚度,但对肺泡上皮细胞的凋亡指数和凋亡蛋白Bcl2、BAX mRNA的表达水平没有明显影响。结论在LPS导致的ALI模型中,VEGF使肺泡上皮细胞紧密连接松解、呼吸膜通透性升高、血浆蛋白渗漏增加、呼吸膜充血、水肿、增厚,导致气体交换障碍。sFlt-1能阻断VEGF的作用,有效维护呼吸膜的正常形态和功能,改善ALI的症状。结论1.本实验采用气管内滴入LPS,成功建立了ALI动物模型。2.在LPS吸入导致的ALI模型中,血浆、BALF和肺组织中的VEGF升高,BALF中VEGF与蛋白定量呈正比,提示VEGF升高参与了LPS所致ALI的病理生理过程。3.采用sFlt-1进行抗VEGF治疗能有效改善ALI导致的低氧、二氧化碳潴留和酸中毒,降低BALF蛋白含量和肺损伤评分。4. VEGF升高使肺泡上皮细胞紧密连接松解,呼吸膜通透性增加,导致了血浆蛋白渗漏,呼吸膜增厚和气体交换障碍。5.sFlt-1治疗ALI的机理在于减轻了VEGF导致的肺泡上皮细胞紧密连接松解,维持呼吸膜正常的形态和功能,减少ALI导致的水和蛋白的渗出。更多还原

【Abstract】 Part I The expression of vascular endothelial growth factor in endotoxin-induced acute lung injury modelObjective To investigate the expression of vascular endothelial growth factor in endotoxin-induced acute lung injury model. Methods Thirty male BALB/c mice were randomly assigned to one of the six groups:LPS 4h group, LPS 24h group, LPS 48h group, PBS 4h group, PBS 24h group, and PBS 48h group. All animals were anesthetized by pentobarbital sodium. LPS (3 mg/kg) was intratracheally instilled via tracheal tube through the trachea to the mice in the LPS group. Mice in the PBS group were instilled an equal volume of PBS as control. Blood gas analysis and vascular endothelial growth factor (VEGF) were measured at predefined time points. Pathohistological change, VEGF and total protein levels in bronchoalveolar lavage fluid were measured. Result Endotoxin administration resulted in an elevation of VEGF (12.44±4.05 pg/ml vs 31.53±1.96 pg/ml, P<0.05) associated with hypoxia (97.27±8.96 mmHg vs 64.24±10.53 mmHg, P< 0.001), carbon dioxide accumulation (37.83±5.75 mmHg vs 49.00±9.19 mmHg, P< 0.05), pulmonary edema, an increase of total protein (183.28±15.58μg/ml vs 569.17±114.68μg/ml, P< 0.05) and VEGF (32.45±6.57 pg/ml vs 160.14±34.25 pg/ml, P< 0.05) in bronchoalveolar lavage fluid. Total protein significantly correlated with VEGF in bronchoalveolar lavage fluid (r=0.676, P< 0.001). The extent of histological abnormalities as assessed by lung injury score peaked at 24 h after LPS challenge.Conclusion Our research indicated that increased VEGF expression may be implicated in the pathogenesis of endotoxin-induced acute lung injury model. Part II Protective effects of anti-vascular endothelial growth factor therapy in endotoxin-induced acute lung injury modelObjective To explore the effect of intervention with anti-vascular endothelial growth factor in endotoxin-induced acute lung injury (ALI) model. Methods Sixty mice were randomly assigned to the PBS group (n=15), LPS group (n=15), sFlt group (n=15), and LPS+sFlt group (n=15). Mice in the four groups were then subdivided into three subgroups (4 h,24 h, and 48 h groups, n=5), respectively. All animals were anesthetized by pentobarbital sodium. LPS (3 mg/kg) was intratracheally instilled via tracheal tube through the trachea to the mice in the LPS group and LPS+sFlt group. Mice in the PBS group and sFlt group were instilled an equal volume of PBS as control. Then sFlt-1(50μg/kg) was infused via tail vein to the mice in the sFlt and LPS+sFlt group. The mice in the PBS and LPS groups were given an equal volume of PBS as control. At predefined time points blood gas analysis, pathohistological change, VEGF and total protein levels in bronchoalveolar lavage fluid were measured. Result Anti-VEGF therapy with sFlt-1 attenuated the hypoxia (64.24±10.53 mmHg vs 84.31±7.23 mmHg, P<0.001), carbon dioxide accumulation (49.00±9.19 mmHg vs 36.51±4.83 mmHg, P< 0.05), and total protein (569.17±114.68μg/ml vs 264.82±76.92μg/ml, P< 0.05) in bronchoalveolar lavage fluid. sFlt-1 administration did not change the level of VEGF in plasma (31.53±1.96 pg/ml vs 39.69±8.98 pg/ml, P>0.05) and BALF (160.14±34.25 pg/ml vs 127.52±17.83 pg/ml, P>0.05). Conclusion Anti-VEGF therapy with sFlt-1 improves the blood gas analysis and attenuates pathohistological development of ALI. sFlt-1 administration might attenuate the thickness of the alveolar-capillary membrane. Part III Protective effects and mechanisms of anti-vascular endothelialgrowth factor therapy in endotoxin-induced acute lung injury modelObjective To investigate the possible mechanism of anti-VEGF therapy in endotoxin-induced acute lung injury (ALI) model. Methods Sixty mice were randomly assigned to the PBS group (n=15), LPS group (n=15), sFlt group (n=15), and LPS+sFlt group (n=15). Mice in the four groups were then subdivided into three subgroups (4 h,24 h, and 48 h groups, n=5), respectively. All animals were anesthetized by pentobarbital sodium. LPS (3 mg/kg) was intratracheally instilled via tracheal tube through the trachea to the mice in the LPS group and LPS+sFlt group. Mice in the PBS group and sFlt group were instilled an equal volume of PBS.Then sFlt-1 (50μg/kg) was infused via tail vein to the mice in the sFlt and LPS+sFlt group. The mice in the PBS and LPS groups were given an equal volume of PBS. At predefined time points, pathohistology of alveolar epithelial barrier, apoptosis index, and the anti-apoptotic protein mRNA express were studied. Result PBS and sFlt group showed intact alveolar-capillary membrane, whereas LPS challenge caused the solution of tight junction between alveolar epithelial cells, type I alveolar epithelial cell shrinkage and detachment from the basement membrane, thickness of the alveolar-capillary membrane and infiltration of cellulose into alveolar spaces. sFlt-1 administration attenuated the solution of tight junction and the thickness of the alveolar-capillary membrane in LPS+sFlt group, but sFlt-1 did not increased the alveoli epithelial cells apoptosis index. Conclusion VEGF causes an increase in the permeability of the lung by breaking lung alveolar-capillary barrier. Anti-VEGF therapy improves the blood gas analysis and attenuates pathohistological development of ALI by decreasing the permeability of alveolar epithelial barrier. Conclusion1. LPS-induced ALI is associated with increased mRNA expression of VEGF in lung tissue and elevated levels of VEGF in blood, lung, and BALF.2. Increased VEGF causes an increase in the permeability of the lung by breaking lung alveolar-capillary barrier.3. Anti-VEGF therapy improves the blood gas analysis and attenuates pathohistological development of ALI.4. Reducing VEGF by treatment with sFlt-1 attenuates the pulmonary edema by decreasing decreasing the permeability of alveolar epithelial barrier.更多还原