乌司他丁对脓毒症大鼠肺微血管通透性及肺组织VEGF表达的影响(1)
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【摘要】 目的 观察乌司他丁注射液对脓毒症大鼠肺微血管通透性及肺组织血管内皮生长因子(VEGF)表达的影响,探讨乌司他丁对脓毒症大鼠肺保护的作用机制。方法 30只健康的雄性SD大鼠随机分为3组:假手术组(A组)、阳性对照组(B组)、乌司他丁治疗组(C组),每组10例。除假手术组外,其他组均施行盲肠结扎穿孔术(CLP)复制大鼠脓毒症模型,治疗组同时给予乌司他丁腹腔注射(100 ku/kg),术后4 h随机处死大鼠5只取组织标本,剩余5只大鼠经颈外静脉注射伊文思蓝观察肺微血管通透性。分别采用逆转录聚合酶链反应(RT-PCR)法和酶联免疫吸附(ELISA)法检测肺组织中VEGF的表达。结果 B组肺微血管通透性及肺组织VEGF表达水平明显高于A、C组;C组肺微血管通透性及肺组织VEGF表达水平高于A组(P<0.05)。结论 乌司他丁能明显抑制脓毒症大鼠肺微血管通透性增高,这种肺保护作用的机制可能与降低VEGF表达有关。
【关键词】乌司他丁注射液;脓毒症;肺微血管通透性;VEGF
Effect of Ulinastatin injection on the permeability of pulmonary microvascular and expression of vascular endothelial growth factor in lung tissue of sepsis in rats
CAI Shi-xia, DENG Lie-hua,TONG Lin, et al.Intensive Care Unit, Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001,China
【Abstract】 Objective To observe the permeability of pulmonary microvascular and expression of vascular endothelial growth factor in septic rats treated with Ulinastatin injection, and to investigate the action mechanism of Ulinastatin in pulmonary function protect.Methods A total of 30 healthy male SD rats were randomly divided into three groups, and each group had 10 rats,Group A received sham operation and the others underwent cecal ligation and puncture (CLP) operation.All the rats were accepted intraperitoneal injection of normal saline ( group A,B) or Ulinastatin (100 ku/kg).After four hours, Lung tissue express of VEGF in all rats were detected by reverse transcription-polymerase chain reaction(RT-PCR) and Enzyme-linked immunosorbent assay(ELISA) methods respectively and the permeability of pulmonary vascular were observed by injecting Evans Blue into jugular vein of rats.Results The permeability of pulmonary microvascular and the expression levels of VEGF in group B were significantly higher than those in group A and C,which in group C were significantly higher than that in group A.Conclusions Ulinastatinis a reliable protection agent for septic induce acute lung injury, and its mechanism of action may be involved in the inhibition of VEGF expression.
【Key words】 Ulinastatin injection;Sepsis;Pulmonary microvascular;VEGF
脓毒症(sepsis)是合并感染的全身性炎症反应综合征(systemic inflammation response syndrome, SIRS),进一步发展可导致多器官功能障碍综合征(MODS),其中呼吸功能障碍发生率较高,出现时间早,其主要的病理生理改变为肺毛细血管通透性增加。血管内皮生长因子(VEGF)是目前已知的最强烈的血管渗透剂。虽然临床研究证实乌司他丁有明确的肺保护作用,但其作用机制仍有待研究。本研究采用盲肠结扎穿孔术(CLP)复制大鼠脓毒症模型观察乌司他丁对脓毒症大鼠肺微血管通透性和VEGF影响,探讨乌司他丁对脓毒症大鼠肺微血管通透性影响及其作用机制。
1 材料与方法
1.1 动物实验分组 SPF级SD雄性大鼠30只,体质量250～300 g(由广东医学院动物中心提供),按随机数字表法分为三组:假手术组(A组),无菌状态下开腹寻找到盲肠后再将其还纳腹腔,然后逐层关腹,术后立即腹腔注射生理盐水(NS)2 ml;CLP组(B组),CLP后立即腹腔注射NS2 ml;乌司他丁(UTI)治疗组(C组), CLP后立即腹腔注射UTI20 kU/kg,加NS至2 ml。术后4 h随机选取5只颈外静脉注射伊文思蓝液,剩余5只大鼠活杀取肺组织标本。为避免伊文思蓝影响肺组织VEGF表达测定,随机各取5只大鼠实验(n=5)。
1.2 CLP模型制备方法[2]动物在乙醚吸入麻醉下无菌开腹手术,在距盲肠末端1 cm处用7号丝线结扎盲肠,用22G针头在被结扎盲肠上对穿3次,注意避免粪便污染,并用生理盐水2 ml湿润肠管,按生理位置还纳肠管。术毕逐层关腹,并于腹壁皮下注射林格氏液50 ml/kg抗休克处理。术后自由进食食水。
1.3 观察指标及标本处理 观察大鼠术后活动、进食、竖毛、腹泻、眼球凹陷、呼吸以及活杀后腹腔肠管扩张、充血、腹水、病灶包裹、肝肿大、肺表面充血等情况。活杀取标本时大鼠在乙醚吸入麻醉下,开腹留取右叶肺缘组织100 mg,用磷酸盐缓冲液(PBS)冲洗后立即置液氮冻存,置-80℃冰箱待逆转录-聚合酶链反应( RT-PCR )检测肺组织VEGFmRNA表达。取右下肺组织200 mg,快速PBS漂洗两次后,置碾钵中用液氮制备肺组织匀浆, 采用酶联免疫吸附ELISA法检测肺组织VEGF蛋白表达。
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