【作者】 张航；

【导师】 张春芳；

【作者基本信息】 中南大学 ， 外科学， 2010， 硕士

【摘要】 目的探讨Bay K8644预处理对家兔肺缺血再灌注损伤的保护作用及其机制。方法40只健康成年清洁级大白兔随机分为4组,每组10只。Ⅰ组：假手术组(Sham组)；Ⅱ组：缺血再灌注组(I瓜组)；Ⅲ组：Bay K8644预处理组(Bay K8644组)；Ⅳ组：缺血预处理组(IP组)。采用在体肺缺血再灌注损伤模型,对比观察各组肺湿／干重比(W／D)、血清及肺组织超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量、肺组织髓过氧化物酶(MPO)含量和肺组织光镜下形态学变化、肺组织电镜下超微结构改变以及肺组织中肺泡表面活性物质相关蛋白-A(SP-A)的表达水平。结果1.各组肺W／D变化：Sham组、Bay K8644组、IP组的肺W／D明显低于I／R组,Sham组、Bay K8644组、IP组与I／R组差异均有统计学意义(P<0.05); BayK8644组与IP组的肺W／D差异无统计学意义(P>0.05)。2.肺组织中SOD活性和MDA、MPO含量的测定：Sham组、Bay K8644组和IP组的肺组织中SOD活性明显高于I／R组,MDA含量明显低于I／R组,MPO含量明显低于I／R组,Sham组、Bay K8644组和IP组与I／R组差异均有统计学意义(P<0.05)；Bay K8644组和IP组差异无统计学意义(P>0.05)。3.血清中SOD活性和MDA含量的测定：组内比较：Sham组随观察实验时间延长SOD活性轻度下调,MDA含量轻度上升,均无统计学意义(P>0.05)；I／R组、Bay K8644组和IP组随再灌注时间延长SOD活性明显降低,MDA含量明显上升,均较缺血前差异显著(P<0.05)。组间比较：缺血前Bay K8644组SOD活性较Sham组及I／R组有轻度下降,而MDA含量有轻度上升,但无统计学差异(P>0.05)；与Sham组比较,Bay K8644组、I／R组和IP组随再灌注时间的延长,各相应时间点SOD活力逐渐下降,而各相应时间点MDA含量逐渐上升,均有统计学差异(P<0.05)；与I／R组比较,Bay K8644组和IP组SOD活力下降程度较低,MDA含量上升程度不高,缺血后各时间点比较有明显差异(P<0.05); Bay K8644组血清中SOD活性和MDA含量与IP组各时间点比较无明显差异(P>0.05)。4.肺组织光镜下形态学变化：Sham组：肺间质及肺泡结构完整,无明显受损,未见明显红细胞渗出。I／R组：肺组织呈病理弥漫性改变,肺不张,肺间质增宽水肿,大量炎症细胞浸润,肺泡壁充血水肿、增厚、炎细胞浸润,肺泡腔内大量红细胞渗出,毛细血管充血水肿,血管壁增厚,血管内大量炎性细胞浸润,渗出严重,肺组织损伤明显。IP组、Bay K8644组：两组基本病理表现相似,肺间质水肿程度较轻,炎症细胞浸润较少,肺泡内红细胞渗出不明显,肺泡结构基本完整,损伤较I／R组明显减轻。5.肺组织电镜下超微结构变化：Sham组：肺泡结构基本正常。Ⅱ型肺泡上皮细胞结构完整,核膜连接紧密,线粒体内质网等细胞器未见异常,微绒毛多且密。部分肺泡毛细血管充血,淤血,毛细血管内皮细胞未见明显异常。I／R组：肺泡隔明显增厚、水肿。Ⅱ型肺泡上皮细胞微绒毛缺失,板层小体大量排空,上皮颗粒全脱失,胶原纤维生成,线粒体等细胞器大量空泡化,细胞核固缩并成凋亡样改变。肺毛细血管内皮细胞肿胀,基底膜增厚,毛细血管淤血,充血,闭锁,狭窄,炎性细胞浸润,间质大量增多。IP组、Bay K8644组：肺泡隔结构轻度增厚,Ⅰ型上皮细胞内仅个别线粒体水肿。Ⅱ型肺泡上皮细胞表面有微绒毛,但稀少,板层小体存在,上皮部分脱颗粒,部分线粒体空泡化。毛细血管腔内炎性细胞滞留,淤血、充血较轻,偶见肺组织炎性细胞浸润,间质轻微水肿。6.免疫组织化学方法检测SP-A表达水平：SP-A免疫组织化学阳性染色呈棕黄色。Sham组、IP组、BayK8644组兔肺SP-A主要以膜状形式连续分布于肺泡腔表面,部分肺泡上皮细胞浆内也可见到棕色颗粒,Sham组肺泡基本完整,BayK8644组、IP组肺泡融合较轻。I／R组肺泡腔内很少见到膜状显色,多以颗粒状染色聚集于肺泡腔内或沉积于肺泡表面,肺泡融合严重。Sham组、IP组、Bay K8644组SP-A表达均强于I／R组,有统计学差异(P<0.05)。Bay K8644组与IP组无明显统计学差异(P>0.05)。结论对于预处理时期有针对性地使用钙通道激动剂Bay K8644增加钙离子内流,对在体兔肺缺血再灌注损伤有一定的保护作用,可模拟IP的肺保护作用。更多还原

【Abstract】 ObjectivesInvestigate the protective effect and mechanism of the preconditioning with calcium channel agonist Bay K8644 of the lungs against ischemia-reperfusion injury in rabbits.MethodsFourty rabbits were divided into four groups randomly: Sham-operated group (Sham, n=10),Ischemic-reperfusion injury group (I/R,n=10), Ischemic preconditioning group(IP,n=10), Bay K8644 preconditioning group (Bay K8644, n=10). Using in vivo model of lung ischemia-reperfusion injury,after test we detected lung W/D changes,lung tissue activities of SOD,lung tissue MPO content,lung tissue MDA content,serum activities of SOD and serum MDA content,also observed morphological changes and ultrastructural changes of lung tissue and expression of SP-A.Results1.The lung W/D changes:After reperfusion,compared with I/R group,the W/D of Sham group、Bay K8644 group and IP group was lower significantly,Sham group、Bay K8644 group、IP group and the I/R group difference was statistically significant (P<0.05);compared with IP group,the Bay K8644 group showed no significant difference on the index(P>0.05).2.Lung tissue SOD activity and MDA,MPO content:Sham group, Bay K8644 group and the IP group of the lung tissue SOD activity was significantly higher than the I/R group,MDA content was lower than the I/R group,MPO content was significantly lower than the I/R group, Sham group、Bay K8644 group、IP group and the I/R group difference was statistically significant (P<0.05);Bay K8644 group and the IP group difference was not statistically significant (P> 0.05).3.Serum SOD activity and MDA content:Within groups:With the experimental observation of prolonged in the Sham group,there was slightly lower in SOD activity and mild increase in MDA content, not statistically significant (P> 0.05).However,the I/R group、Bay K8644 group and IP group with prolongation of reperfusion,SOD activity was significantly decreased,MDA content was increased,compared with before ischemia significantly different (P<0.05).Between groups:SOD activity before ischemia Bay K8644 group compared with Sham group and I/R group decreased slightly,while the MDA content increased slightly,but no significant difference (P> 0.05);Compared with the Sham group,Bay K8644 group、I/R group and the IP group with reperfusion time prolonged,the corresponding time points SOD activity decreased gradually,while the corresponding time points MDA content increased gradually,were statistically significant (P <0.05);Compared with the I/R group,Bay K8644 group and the IP group decreased less SOD activity,MDA content increased level was not high,compared at different time points after ischemia,were significantly different (P<0.05);Compared with IP group,there was no significant difference at different corresponding time points in the serum SOD activity and MDA content (P>0.05).4.Lung tissue under light microscope morphological changes:Sham group:Pulmonary interstitial and alveolar structural integrity, no significant damage,no significant red blood cell leakage.I/R group:Athological lung tissue showed diffuse changes,atelectasis,pulmonary interstitial edema widened,a large number of inflammatory cell infiltration,alveolar wall congestion and edema,thickening,inflammatory cell infiltration,alveolar exudate a large number of red blood cells,capillary congestion and edema,vascular wall thickening,vascular and inflammatory cell infiltration,exudation serious,lung injury significantly.IP group and Bay K8644 group:Two groups were similar to the basic pathology,pulmonary interstitial edema to a lesser extent,less infiltration of inflammatory cells,red blood cells within the alveolar exudate was not obvious,the basic integrity of alveolar structure,damage than the I/R group was significantly reduced.5.Lung tissue under electron microscopy ultrastructural changes: Sham group:Normal alveolar structure.TypeⅡalveolar epithelial cell structural integrity,membrane connection close,mitochondria and endoplasmic reticulum and other organelles were normal,large and dense microvilli.Part of the alveolar capillary congestion,capillary endothelial cells had no obvious abnormalities.I/R group:Thickened alveolar septa and edema.TypeⅡalveolar epithelial cells lack microvilli,a large number of empty lamellar bodies,epithelial granules demyelination,collagen fibrils,a large number of mitochondria and other organelles vacuolization,nuclear condensation and apoptotic-like changed into.Pulmonary capillary endothelial cell swellied,basement membrane thickened,capillary congestion,atresia,stenosis,inflammatory cell infiltration and large increase in interstitial.IP group and Bay K8644:Structure slightly thickened alveolar septum.I epithelial cells only individual mitochondrial swelled.TypeⅡalveolar epithelial cell surface microvilli was rare,lamellar bodies existed,there were epithelial part of the degranulation and some vacuolization of mitochondria.Capillary cavity retention of inflammatory cells,congestion,congestion lighter,occasionally lung tissue inflammatory cell infiltration,mild interstitial edema.6.Immunohistochemistry to detect the expression of SP-A:Immunohistochemical staining of SP-A was positive for brown.The SP-A of Sham group,Bay K8644 and IP group in rabbits lung,mainly in the form of a continuous distribution of membranous surface in the alveolar space,part of the cytoplasm of alveolar epithelial cells can also be seen brown granules.Alveolars of Sham group were basic integrity,however,less alveolar fusion in the Bay K8644 group and IP group.I/R group alveolar membranous rarely seen color,granular staining mostly gathered in the alveolar cavity or deposited in the alveolar surface,severe alveolar fusion.Sham group,IP group,Bay K8644 group of SP-A expression was respectively stronger than I/R group,there was significant difference (P<0.05).Bay K8644 group and the IP group was no significant difference (P> 0.05).ConclusionPreconditioning period for the targeted use of appropriate calcium channel agonist Bay K8644 increased transient calcium influx, which had protective effects on lung ischemia-reperfusion and could simulate the endogenous protective effects of IP in the vivo rabbit I/R injury model.更多还原