骨桥蛋白对内毒素致大鼠急性肺损伤后肺纤维化中细胞因子的影响

  本文关键词：骨桥蛋白对内毒素致大鼠急性肺损伤后肺纤维化中细胞因子的影响，由笔耕文化传播整理发布。

        目的：通过观察骨桥蛋白抗体（Anti-OPN-Ab）对内毒素腹腔注射所致的SD大鼠急性肺损伤后肺纤维化肺组织中核因子-κB（NF-κB）及炎性细胞因子肿瘤坏死因子（TNF-α）、干扰素γ（IFN-γ）、白细胞介素4（IL-4）、白细胞介素10（IL-10）表达水平的影响，探讨骨桥蛋白在急性肺损伤后肺纤维化过程中的作用，进一步阐明肺损伤后纤维化的发病机制。方法：通过腹腔注射内毒素（LPS）建立大鼠急性肺损伤后肺纤维化模型，60只清洁级雌性SD大鼠随机分为3组，空白对照组（n=20，腹腔注射无菌生理盐水NS），内毒素组（n=20，腹腔注射LPS，5mg/kg），干预组（n=20，腹腔注射LPS，5mg/kg，5分钟后再次腹腔注射骨桥蛋白抗体0.5ml，效价为1:32）；在随机分为3组的基础上又按3d、7d、14d、28d不同时间点随机分为4组，在0d、1d、2d连续用药3天，共3次，建立肺损伤后肺纤维化模型。各组大鼠按上述时间点处死，留取标本行相应检测。①取右肺下叶行HE染色及Masson染色观察肺组织病理变化情况，②右肺上叶免疫印迹（Western blotting）检测肺组织匀浆细胞浆与细胞核NF-κB的表达。③双抗体酶联免疫吸附法（ELLISA）测定大鼠左肺肺组织匀浆炎症细胞因子TNF-α、IFN-γ、IL-4、IL-10的表达。结果：1、NS组大鼠肺组织HE染色光镜下肺泡结构正常，无破坏，无炎性渗出物，肺间隔无增宽及炎性细胞浸润；内毒素组于3d、7d组均有较重的炎症反应即肺泡结构的破坏，肺泡腔闭陷融合，肺泡间隔断裂，炎性细胞填充于肺泡腔及肺间质，并伴有出血、水肿，蓝色胶原纤维沉积在炎症较重的部位；以后炎症反应逐渐减轻，而纤维化程度越来越重，干预组上述病理表现均较内毒素组减轻。在整个实验过程中内毒素组肺泡炎评分均较NS组明显升高（p<0.01,p<0.05）,炎症高峰时期在3d、7d，干预组肺泡炎程度明显较内毒素组下降（p<0.01），，于28d时与NS组比较差异无统计学意义（p>0.05）；内毒素组与干预组肺纤维化评分均较NS组升高（p<0.05，p<0.01），但干预组肺纤维化程度更低，与内毒素比较差异有意义（p<0.05,p<0.01）。2、内毒素组及干预组大鼠肺组织匀浆细胞浆及细胞核NF-κB的表达在3d、7d、14d、28d与NS组比较均有明显的统计学差异（p<0.01，p<0.05），在3d、7d组达峰；内毒素组与干预组比较，细胞浆NF-κB的表达无差异(p>0.05)，细胞核NF-κB的表达有统计学差异（p<0.01）。3、内毒素组及干预组大鼠肺组织匀浆致炎因子TNF-α、IFN-γ与NS组比较两组存在统计学差异（p<0.05，p<0.01），表达最高峰3d、7d组，干预组较内毒素组有明显下降（p<0.05,p<0.01）；内毒素组及干预组抗炎因子IL-4、IL-10较NS组有明显升高（p<0.05，p<0.01），内毒素升高更为明显（p<0.05）。结论：多次小量腹腔注射LPS能够成功复制急性肺损伤后肺纤维化模型，损伤最重的时期在3d与7d，纤维化最明显的时刻在28d。内毒素组肺组织在早期就开始有NF-κB及炎症因子TNF-α、IFN-γ、IL-4、IL-10的升高，表达高峰时间在3d与7d，以后随时间的推移表达量逐渐下降。骨桥蛋白可以通过促进NF-κB通路激活，上调致炎因子TNF-α、IFN-γ，下调抗炎因子IL-4、IL-10，参与急性肺损伤后肺纤维的过程。使用一定剂量骨桥蛋白抗体可以一定程度上减弱肺损伤后肺纤维化。

    Objective:For investigating the role of osteopontin and futherpathogenesis in acute lung injuried lung fibrosis,we observe the nuclearfactor kappaB （NF-κB） and IFNlammatory cellscytokines expressionlevels,such as tumor necrosis factor （TNF-α）， interferon gamma（IFN-γ）,Interleukin-4（IL-4）,Interleukin-10（IL-10）in SD rats’lungtissue after intraperitoeally with lipopolysaccharid（eLPS）and osteopontinantibody.Methods:Acute lung injuried lung fibrosis SD rats model wasestablished by intraperitonesl injection with LPS.60femaleSprague-Dawley rats were randomly divided into three groups, controlgroup (n=20,the intraperitoneal injection of sterilesaline the NS),endotoxin model group (n=20,the intraperitoneal injection of LPS,5mg/kg), the intervention group (n=20, the intraperitoneal injection ofLPS,5mg/kg, intraperitoneal injection of osteopontin antibody0.5mltiter1:32after5minutes), Also randomly divided into for groups based ondifferent time points3d,7d,14d,28d after randomly divided into threegroups. three groups were established lung fibrosis model by injectiondrug three times., Every rats in each group were sacrificed on above pointin time,and specimens for corresponding detection.①observed lungtissue pathological changes by HE staining and Masson staining withright lower lung leaves②observed nucleus of NF-κB expression in lung tissue by immunoblot (Western blot) with right upper lung leaves③observed IFNlammatory cytokines expression level such as TNF-alpha,IFN-γ, IL-4, IL-10by enzyme-linked immunosorbent assay (ELLISA)with rats left lung tissue Results:1, The pathology was observed underHE staining and Masson staining200times microscope NS grouprats’lung tissue were normal, no destruction, no IFNlammatory, nopulmonary interval broadened and IFNlammatory cell IFNiltration; whileendotoxin group the alveolar architecture and alveolar septum weredestructed, alveolar spaces closed and integration IFNlammatory cellsfilled the alveolar spaces and pulmonary interstitium, accompaniedbleeding, edema,blue collagen fibers began to deposited in the heavierparts at3d and7d groups;Later,the inflammatory response weaken butmore blue collagen fibersthe appeared at injuried site of the lung.Compared with endotoxin group the intervention group pathologicalmanifestations were weaken during the whole experiment, comparedwith the NS group endotoxin group alveolitis score was significantlyincreased (p<0.01, p<0.05) the peak period of inflammation in the3d,7d,while the intervention group was significantly more decreased (p<0.01)compared with NS group, the difference was not statistically significant(p>0.05) at28days Endotoxin group and intervention group, lungfibrosis score increased (p<0.05, p<0.01) than those in NS group but thedegree of pulmonary fibrosis in intervention group was lower than endotoxin group (p<0.05,p<0.01)2,compared with NS group thecytoplasm and nucleus of NF-κB expression in rat lung tissuehomogenates began to rise in the early time,which was statisticallysignificant (p<0.01) and the3days7days groups were most obviousbetween model group and intervention group.cytoplasm of NF-κBexpression, including no difference between the toxin model group andintervention group (p>0.05), while the nucleus of NF-κB expressionbetween the two groups which was statistically significant (p<0.01).3,The proIFNlammatory cytokines expression level such as TNF-alpha,IFN-γ increased at an early stage and the amount existed consistency withthe nucleus of NF-kappa B still8h and7d groups is the most obviousbetween endotoxin model group and intervention group in lunghomogenates At the same time the two groups compared with NS groupthere was significant difference (p<0.05, p<0.01), later,theIFNlammatory factor expression gradually decreased, the quantity ofproIFNlammatory cytokines TNF-alpha and IFN-γ expression in theintervention group creased significantly than model group(p<0.05,p<0.01); while anti-IFNlammatory cytokines IL-4, IL-10in the modelgroup and intervention group remained significantly higher (p<0.05,p<0.01),compared with NS group also intervention group increased moresignificantly (p<0.05)compared with model group.Conclusion:multiplesmall intraperitoneal injection with LPS can copy acute lung injury and lung fibrosis model, the most serious injury period is in8h and7d, themost significant fibrosis moment is in28d. The expression of NF-κB andIFNlammation of TNF-alpha, IFN-γ, IL-4, IL-10increased in the earlytime in endotoxin model of lung tissue and the peak is3d，7d after theexpression level was gradually decline over time pulmonary fibrosisassociated with NF-κB and inflammation of TNF-alpha and IFN-γ, IL-4,IL-10Osteopontin involved in the process of pulmonary fibrosis byactiving NF-κB pathway, increasing of proinflammatorycytokines,decreasing the anti-inflammatory factor.The use of a certaindose of osteopontin antibody can weaken this fibrosis at certain extent.

骨桥蛋白对内毒素致大鼠急性肺损伤后肺纤维化中细胞因子的影响

摘要4-7Abstract7-10前言11-14材料与方法14-20结果20-30讨论30-39结论39-40参考文献40-45英汉缩略词对照表45-46致谢46-47综述47-60    参考文献55-60

  本文关键词：骨桥蛋白对内毒素致大鼠急性肺损伤后肺纤维化中细胞因子的影响，由笔耕文化传播整理发布。