本文關(guān)鍵詞：骨橋蛋白對內(nèi)毒素致大鼠急性肺損傷后肺纖維化中細胞因子的影響，由筆耕文化傳播整理發(fā)布。

        目的：通過觀察骨橋蛋白抗體（Anti-OPN-Ab）對內(nèi)毒素腹腔注射所致的SD大鼠急性肺損傷后肺纖維化肺組織中核因子-κB（NF-κB）及炎性細胞因子腫瘤壞死因子（TNF-α）、干擾素γ（IFN-γ）、白細胞介素4（IL-4）、白細胞介素10（IL-10）表達水平的影響，探討骨橋蛋白在急性肺損傷后肺纖維化過程中的作用，進一步闡明肺損傷后纖維化的發(fā)病機制。方法：通過腹腔注射內(nèi)毒素（LPS）建立大鼠急性肺損傷后肺纖維化模型，60只清潔級雌性SD大鼠隨機分為3組，空白對照組（n=20，腹腔注射無菌生理鹽水NS），內(nèi)毒素組（n=20，腹腔注射LPS，5mg/kg），干預(yù)組（n=20，腹腔注射LPS，5mg/kg，5分鐘后再次腹腔注射骨橋蛋白抗體0.5ml，效價為1:32）；在隨機分為3組的基礎(chǔ)上又按3d、7d、14d、28d不同時間點隨機分為4組，在0d、1d、2d連續(xù)用藥3天，共3次，建立肺損傷后肺纖維化模型。各組大鼠按上述時間點處死，留取標本行相應(yīng)檢測。①取右肺下葉行HE染色及Masson染色觀察肺組織病理變化情況，②右肺上葉免疫印跡（Western blotting）檢測肺組織勻漿細胞漿與細胞核NF-κB的表達。③雙抗體酶聯(lián)免疫吸附法（ELLISA）測定大鼠左肺肺組織勻漿炎癥細胞因子TNF-α、IFN-γ、IL-4、IL-10的表達。結(jié)果：1、NS組大鼠肺組織HE染色光鏡下肺泡結(jié)構(gòu)正常，無破壞，無炎性滲出物，肺間隔無增寬及炎性細胞浸潤；內(nèi)毒素組于3d、7d組均有較重的炎癥反應(yīng)即肺泡結(jié)構(gòu)的破壞，肺泡腔閉陷融合，肺泡間隔斷裂，炎性細胞填充于肺泡腔及肺間質(zhì)，并伴有出血、水腫，藍色膠原纖維沉積在炎癥較重的部位；以后炎癥反應(yīng)逐漸減輕，而纖維化程度越來越重，干預(yù)組上述病理表現(xiàn)均較內(nèi)毒素組減輕。在整個實驗過程中內(nèi)毒素組肺泡炎評分均較NS組明顯升高（p<0.01,p<0.05）,炎癥高峰時期在3d、7d，干預(yù)組肺泡炎程度明顯較內(nèi)毒素組下降（p<0.01），，于28d時與NS組比較差異無統(tǒng)計學(xué)意義（p>0.05）；內(nèi)毒素組與干預(yù)組肺纖維化評分均較NS組升高（p<0.05，p<0.01），但干預(yù)組肺纖維化程度更低，與內(nèi)毒素比較差異有意義（p<0.05,p<0.01）。2、內(nèi)毒素組及干預(yù)組大鼠肺組織勻漿細胞漿及細胞核NF-κB的表達在3d、7d、14d、28d與NS組比較均有明顯的統(tǒng)計學(xué)差異（p<0.01，p<0.05），在3d、7d組達峰；內(nèi)毒素組與干預(yù)組比較，細胞漿NF-κB的表達無差異(p>0.05)，細胞核NF-κB的表達有統(tǒng)計學(xué)差異（p<0.01）。3、內(nèi)毒素組及干預(yù)組大鼠肺組織勻漿致炎因子TNF-α、IFN-γ與NS組比較兩組存在統(tǒng)計學(xué)差異（p<0.05，p<0.01），表達最高峰3d、7d組，干預(yù)組較內(nèi)毒素組有明顯下降（p<0.05,p<0.01）；內(nèi)毒素組及干預(yù)組抗炎因子IL-4、IL-10較NS組有明顯升高（p<0.05，p<0.01），內(nèi)毒素升高更為明顯（p<0.05）。結(jié)論：多次小量腹腔注射LPS能夠成功復(fù)制急性肺損傷后肺纖維化模型，損傷最重的時期在3d與7d，纖維化最明顯的時刻在28d。內(nèi)毒素組肺組織在早期就開始有NF-κB及炎癥因子TNF-α、IFN-γ、IL-4、IL-10的升高，表達高峰時間在3d與7d，以后隨時間的推移表達量逐漸下降。骨橋蛋白可以通過促進NF-κB通路激活，上調(diào)致炎因子TNF-α、IFN-γ，下調(diào)抗炎因子IL-4、IL-10，參與急性肺損傷后肺纖維的過程。使用一定劑量骨橋蛋白抗體可以一定程度上減弱肺損傷后肺纖維化。

    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.

骨橋蛋白對內(nèi)毒素致大鼠急性肺損傷后肺纖維化中細胞因子的影響

摘要4-7Abstract7-10前言11-14材料與方法14-20結(jié)果20-30討論30-39結(jié)論39-40參考文獻40-45英漢縮略詞對照表45-46致謝46-47綜述47-60    參考文獻55-60

  本文關(guān)鍵詞：骨橋蛋白對內(nèi)毒素致大鼠急性肺損傷后肺纖維化中細胞因子的影響，由筆耕文化傳播整理發(fā)布。