【摘要】 目的观察C57BL/6小鼠一次性大剂量(15Gy)胸部照射后肺组织高迁移率族蛋白B1(high mobility group protein,HMGB1)表达和支气管肺泡灌洗液(bron choalveolar lavage fiuid,BALF)中HMGB1水平的改变,探讨HMGB1在小鼠放射性肺损伤中的表达及作用。方法 42只C57BL/6小鼠随机分为对照组(6只)、照射12h组(12只)、照射1周组(12只)和照射4周组(12只),ELEKTA直线加速器一次性15Gy照射小鼠胸部,建立放射性肺损伤模型。在相应的时间点获取BALF并处死照射后及对照组小鼠,保存肺组织标本。采用实时qRT-PCR检测肺组织HMGB1mRNA水平,采用蛋白质印迹法和免疫组化染色分析肺组织HMGB1蛋白水平。BALF中HMGB1、IL-17A、TNF-α和IL-6含量采用ELISA法检测,HE染色后光镜下观察肺组织病理形态变化。结果 HE染色显示,照射1周后肺泡腔内和细支气管壁可见成堆炎细胞浸润,毛细血管充血,肺间质水肿。照射4周后更为明显。HMGB1免疫组化染色显示,照射后肺组织细胞HMGB1表达明显增强。蛋白质印迹结果显示,对照组HMGB1蛋白表达量为0.42±0.10,照射12h组为0.78±0.13,照射1周组为1.23±0.21,照射4周组为1.56±0.22。照射后12h肺组织HMGB1蛋白表达量较对照组明显增高,F=11.809,P=0.035;并随时间延长而进一步升高,P<0.05。RT-PCR结果显示,对照组肺组织HMGB1mRNA相对表达水平(ΔCt)为9.69±1.40,照射12h组为7.58±1.01,照射1周组为5.45±1.06,照射4周组为3.55±0.73。照射12h组ΔCt显著性低于对照组,F=13.410,P=0.034;并随照射后时间延长肺组织HMGB1mRNA的ΔCt值进一步降低,P<0.05。ELISA结果显示,小鼠肺照射后各时间点BALF中HMGB1、IL-17A、TNF-α和IL-6水平均明显升高,P<0.01。结论小鼠肺照射后HMGB1表达改变,且存在时间依赖性;HMGB1参与放射性肺损伤过程。更多还原

【Abstract】 OBJECTIVE To investigate the changes of HMGB1 expression in lung tissue and HMGB1 level in BALF after total thoracic irradiation in mice.METHODS Forty-two mice were randomly divided into control group(n=6),post-irradiation 12hgroup(n=12),1week post-irradiation group(n=12)and 4weeks post-irradiation group(n=12).The total chest of mice were irradiated at the dose of 15 Gy one time by ELEKTA linear accelerator to establish radiation-induced lung injury model.The expression of HMGB1 mRNA in lung tissue was quantitatively detected by real-time RT-PCR and HMGB1 protein levels by Western blot and immunohistochemistry staining.HMGB1,TNF-α,IL-17 Aand IL-6in BALF were detected by ELISA.RESULTS HE staining showed that bronchioles visible piles of inflammatory cell infiltration,capillary congestion,and interstitial edema occurred in lung tiusses after 1 week post-irradiation,and the damages were more apparent after 4weeks post-irradiation.HMGB1 immunohistochemical staining showed that HMGB1 expression in lung tissue irradiated was significantly enhanced.Westernblot results showed HMGB1/β-actin grayscale ratios were 0.42±0.10 in control group,0.78±0.13 in irradiated 12 hgroup,1.23±0.21 in 1wpost-irradiation group,and 1.56±0.22 in 4wpost-irradiation group.HMGB1 protein levels in lung tissue in 12hpost-irradiation group was significantly higher than that in control group,F=11.809,P=0.035,and which was further increased with time(P<0.05).RT-PCR showed that in control group,12hpost-irradiation group,1wand 4wpost-irradiation groups,HMGB1 mRNA relative expression levels(ΔCt)were 9.69±1.40,7.58±1.01,5.45±1.06,3.55±0.73.HMGB1mRNAΔCt of 12hpost-irradiation group was significantly lower than that of control group,F=13.410,P=0.034,and which was further reduced with the extension of irradiation time(P<0.05).ELISA results showed that HMGB1,IL-17 A,TNF-αand IL-6levels in bronchoalveolar lavage fluid of irradiated mice were significantly higher at each time point than that of control group(P<0.01).CONCLUSION The changes of HMGB1 expression level were significant after irradiation in mice,and HMGB1 may participate in radiation induced lung injury.更多还原