小鼠急性自体血栓性肺栓塞后内皮型一氧化氮合酶及一氧化氮表达变化

Changes of endothelial nitric oxide synthase and nitric oxide level in the experimental mice model of acute pulmonary embolism

目的:建立操作简便、成功率高、耗资低廉的急性血栓性肺栓塞(acute pulmonary thrombo-embolism,APE)小鼠动物模型,并观察肺内内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)及血清一氧化氮(nitric oxide,NO)的表达变化情况。方法:雄性C57BL/6小鼠(n=234),随机分为三组,即正常对照组(n=39)、假手术组(n=66)和模型组(n=129),各组又随机分为1 h、1d和2d三个亚组。模型组自右颈静脉注入自体血栓栓子,假手术组以0.9%氨化钠偏代替自体血栓栓子注入。到指定时间点处死小鼠,取肺组织进行大体观察,苏木素-伊红(HE)染色观察肺组织和肺血管结构的变化,Mallory氏磷钨酸苏木精(PTAH)染色显示血管内血栓中的纤维素。以试剂盒测定外周血NO水平,免疫组化观察肺组织中eNOS蛋白的表达情况。结果:模型组HE染色光镜下可见注入的血栓栓子,PTAH染色光镜下栓子中可见蓝紫色纤维素网状结构,且大多数栓子栓塞于肺动脉段水平。与1 h模型组相比,1 d和2 d模型组的栓子数目逐渐减少(分别约为1 h模型组的90%和30%)。另外,部分远端肺动脉中发现有大量红细胞聚集,可能是由于继发血栓的形成。注栓1 d后,模型组与正常让外周血NO水平(22.666±2.937)μmol/L与正常对照组[(50.596±5.799)μmol/L,P<0.01]和假手术组[(37.797±4.880)μmol/L,P<0.05]比较差异有统计学意义。与正常组及假手术组相比,1 d模型组肺动脉内皮eNOS蛋白表达有所增高。结论:用自体血栓栓子可成功诱导小鼠APE形成,APE后血清NO生成减少,肺动脉内皮eNOS表达升高。

Objective：To find a way to establish the mice model of acute pulmonary embolism（APE） with simple manipulation,high success and feasibility,low cost and similar to human pathophysiology as well,then observe the levels of lung endothelial nitric oxide synthase（eNOS） and blood nitric oxide（NO）.Methods： A total of 234 healthy male C57BL/6 mice were randomly divided into three groups： control group（n=39）,sham group（n=66） and APE group（n=129）.The survival mice were randomly divided into 1 h,1 D and 2 D three sub-groups.After the right jugular veins were isolated,autologous blood thrombi were injected into the veins of mice of APE group while normal saline was injected into the veins of sham group.In appointed time,all the mice were sacrificed and lung tissues of them were evaluated by morphological observation,histological section of hematoxylin-eosin（HE） staining and Mallory＇s phosphotungstic acid hematoxylin（PTAH） staining which is a staining to evidence fibrin in tissues.The plasma level of nitric oxide（NO） of each animal was determined.The level of endothelial nitric-oxide synthase（eNOS） protein in lung tissue was evaluated by immunohistochemical method.Results： In APE group,thrombi could be detected in the histological section of lung tissue and covered with the blue-purple fibrin nest based on PTAH staining,while most of the thrombi obstruct the segment level of the pulmonary artery.However,for the 1D and 2D APE group,the numbers of thrombi were reduced（around 90%,30%thrombi left compared with 1h APE group,respectively）.Furthermore,a large number of erythrocytes could be found to aggregate in distal pulmonary arteries due to the formation of secondary thrombosis.The plasma level of NO in APE group [（22.666±2.937）μmol/L] was significantly lower than that of control group [（50.596±5.799）μmol/L,P0.01] and sham group respectively [（37.797±4.880）μmol/L,P0.05] at 1D after embolization.Compared with control group and sham group,the expression of eNOS protein in APE group was higher at 1D after embolization.Conclusion： The mice model of APE successfully formed with autologous blood thrombi,while decrease of NO biosynthesis and increase of the eNOS protein expression can be observed during APE.