伊洛前列素对博来霉素诱导小鼠肺纤维化的干预作用及相关机制研究

【作者】 刘涌；

【导师】 朱元珏； 高金明；

【作者基本信息】 中国协和医科大学 ， 呼吸内科， 2009， 博士

【摘要】 目的:建立博来霉素诱导小鼠肺纤维化模型,观察伊洛前列素对肺纤维的可能干预作用;并进一步探讨可能的机制。方法:1.应用单次气管内注入博来霉素(2mg/kg体重)的方法复制小鼠肺纤维化模型,并通过单次腹腔内注射伊洛前列素(200ug/kg体重)来实现对小鼠肺纤维化的干预。2.观测小鼠体重变化,生存指数,肺组织羟脯氨酸测定,病理切片Ashcroft评分及肺泡间隔厚度测定评估小鼠肺纤维化的严重程度,采用Flexivent系统评价博来霉素诱导肺纤维化小鼠肺功能改变,并评价伊洛前列素的干预作用。3.通过半定量RT-PCR测定肺组织中细胞因子和趋化因子mRNA的表达水平,评价伊洛前列素对细胞因子表达的影响。4.应用ELISA试剂盒测定BALF中细胞因子和趋化因子在蛋白水平的表达,评价伊洛前列素对细胞因子表达的影响。结果:1.伊洛前列素组小鼠与博来霉素组相比,在如下方面存在显著性差异:气管内注药后第6至第18天平均体重(p＜0.05),生存指数(90%vs 40%,p＜0.01),肺组织羟脯氨酸测定(56.04±2.23 ug/ml vs 77±6.01ug/ml,p＜0.05),肺纤维化Ashcroft评分(3.35±0.54 vs 5.64±0.18,p＜0.01),肺泡间隔厚度测定(23.03±4.91 vs 56.12±4.06,p＜0.01),小鼠肺组织弹性(49.96 cmH20/ml±1.83 vs 86.84±13.13cmH20/ml,p＜0.01),肺组织顺应性(0.02015±0.00074 ml/cmH20 vs 0.01448±0.00178ml/cmH20,p＜0.05),均显示伊洛前列素治疗组优于博来霉素组。2.伊洛前列素组与博来霉素组相比,可显著降低以下细胞因子mRNA表达水平:IL-6(0.75±0.33 vs 1.67±0.22,p＜0.05),TNF-α(0.28±0.02 vs0.44±0.05,p＜0.05),TGF-β(0.90±0.10 vs 1.26±0.07,p＜0.05);并可显著提高以下细胞因子表达水平:IFN-γ(0.90±0.10 vs 1.26±0.07,p＜0.05),CXCL10(1.03±0.09 vs 0.76±0.03,p＜0.05),CXCL11(0.35±0.04 vs 0.22±0.02,p＜0.05)。3.在蛋白水平伊洛前列素可影响BALF中以下细胞因子的表达:降低IL-6(26.5±4.04 pg/ml vs 131.5±38.21pg/ml,p＜0.05)和TGF-β(1906±988 pg/ml vs 14350±4798pg/ml,p＜0.01)表达水平;并提高以下细胞因子的表达:IFN-γ(59.25±10.49 pg/ml vs 18.91±9.84 pg/ml,p＜0.05),CXCL10(108.4±5.46 pg/ml vs 65.88±6.39 pg/ml,p＜0.01)。结论:1.伊洛前列素对博来霉素诱导小鼠肺纤维化具有明显的保护作用,降低死亡率、肺泡炎、肺泡破坏程度及纤维化程度。2.伊洛前列素的保护作用主要是通过抑制IL-6、TNF-α和TGF-β等细胞因子表达。3.另外伊洛前列素可通过提高IFN-γ、CXCL10、CXCL11的表达起保护作用。更多还原

【Abstract】 Objective●To investigate the interventional effectiveness of Iloprost on Bleomycin-induced pulmonary fibrosis in mice.●To further explore the mechanisms of the preventive role of Iloprost on bleomycin-induced pulmonary fibrosis in mice.Method●The BPF model was established by a single dosage of 2mg/kg bleomycin intra-tracheal injection.Single dosage of 200ug/kg Iloprost intraperitoneal injection was given to the mice.●The effectiveness was evaluated by the weight curve and survival rate. The fibrosis degree was determined biochemically by hydroxyproline assay in lung tissue,Ashcroft’ scoring pathologically by using HE and Masson Trichrome stained lung tissue,alveolar septal thickening measurement and physical index.●The expression of the cytokines and chemokines in lung tissue was assayed by semi-quantitative RT-PCR.●ELISA kits was used to evaluate the cytokines and chemokines in BALF at protein level.Result●Compare to the mice administrated by bleomycin,there was significant protection in BPF mice treated with iloprost in several aspects:weight (p＜0.05),survival rate(90%vs 40%,p＜0.01),hydroxyproline content (56.04±2.23 ug/ml vs 77±6.01ug/ml,p＜0.05),Ashcroft’ scoring (3.35±0.54 vs 5.64±0.18,p＜0.01),alveolar septal thickening (23.03±4.91 vs 56.12±4.06,p＜0.01),compliance(0.02015±0.00074 ml/cmH2O vs 0.01448±0.00178ml/cmH2O,p＜0.05) and elastance (49.96±1.83 cmH2O/ml vs 86.84±13.13cmH2O/ml,p＜0.01) of the lung tissue.●The mRNA expression of the cytokines tested,IL-6(0.75±0.33 vs 1.67±0.22,p＜0.05),TNF-α(0.28±0.02 vs 0.44±0.05,p＜0.05),TGF-β(0.90±0.10 vs 1.26±0.07,p＜0.05) was significantly down-regulated in the lung tissues from mice treated with iloprost.The expression of IFN-y (0.90±0.10 vs 1.26±0.07,p＜0.05),CXCL10(1.03±0.09 vs 0.76±0.03, p＜0.05),CXCL11(0.35±0.04 vs 0.22±0.02,p＜0.05) was significantly up-regulated.●The up-regulated expression of IFN-γ(59.25±10.49 vs 18.91±9.84 pg/ml,p＜0.05),CXCL10(108.4±5.46 vs 65.88±6.39 pg/ml,p＜0.01) and down-regulated expression of IL-6(26.5±4.04 vs 131.5±38.21 pg/ml,p＜0.05),TGF-β(1906±988 vs 14350±4798pg/ml,p＜0.01) was found in BALF from mice treated with iloprost.Conclusion●The mortality and pulmonary fibrosis degree were significantly reduced by the intrapedtoneal injection of Iloprost.●The inhibition of inflammatory cytokines expression was the potential mechanism that iloprost protected from bleomycin-induced pulmonary fibrosis in mice.●The induction of anti-angiogenesis mediators associated with upregulation of IFN-γ,including CXCL10 and CXCL11,was another potential protective mechanism of iloprost.更多还原