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目的探讨骨桥蛋白(OPN)在高氧所致小鼠急性肺损伤(ALI)发病过程中的作用及其机制。方法将72只OPN基因野生型(OPN+/+)小鼠随机分为正常对照组(WN组)、高氧24h组(WO1组)、高氧48h组(WO2组)、高氧72h组(WO3组),每组18只;将72只OPN基因缺失型(OPN-/-)小鼠随机分为正常对照组(DN组)、高氧24h组(DO1组)、高氧48h组(DO2组)、高氧72h组(DO3组),每组18只。正常对照小鼠呼吸室内空气,高氧小鼠置于密闭的氧气室(氧浓度>95%)。行支气管肺泡灌洗液(BALF)细胞计数及分类,评价小鼠肺损伤程度;将OPN-/-、OPN+/+小鼠各20只暴露于高氧下纪录生存时间;明胶酶谱分析基质金属蛋白酶2(MMP2)、MMP9的释放及活性;逆转录聚合酶链反应(RT PCR)检测OPN、MMP2、MMP9、金属蛋白酶1组织抑制剂(TIMP1)和TIMP2mRNA的表达。结果DO3组小鼠肺损伤较WO3组更严重。OPN-/-小鼠生存期显著缩短(χ2=23.91、P<0.01)。DO3组小鼠BALF细胞总数[(72.2±22.3)×104/L]显著高于WO3组[(39.7±10.4)×104/L,P<0.05],其中中性粒细胞数增加近8倍[(207.54±36.45)×103/L,(25.33±6.43)×103/L,P<0.01]。明胶酶谱分析显示,DO3组小鼠BALF活化的MMP9表达[(4.36±0.65)×104]显著高于WO3组[(2.84±0.44)×104,P<0.01]。RT PCR显示,WO2、WO3组小鼠肺组织OPNmRNA表达(0.87±0.08、0.92±0.07)显著高于WN组(0.69±0.04,P均<0.05);WO3组小鼠TIMP1表达(1.09±0.12)显著高于DO3组(0.62±0.09,P<0.05);WO2、WO3组小鼠肺组织TIMP2mRNA表达(1.05±0.23、0.99±0.13)分别与DO2、DO3组比较差异均有统计学意义(0.59±0.11、0.75±0.16,P<0.01、<0.05)。结论OPN通过促进TIMP的表达而抑制MMP的释放和激活,从而减轻高氧所致的ALI。
Objective To investigate the role of osteopontin (OPN) in the pathogenesis of hyperoxia-induced acute lung injury (ALI) in mice and its mechanism. Methods 72 untreated OPN + wild type mice were randomly divided into normal control group (WN group), high oxygen group (WO1 group), high oxygen group (group WO2), high oxygen group (group WO3) (OPN - / -) mice were randomly divided into normal control group (DN group), hyperoxia group (DO1 group), hypoxia 48h group (DO2 group) ), Hyperoxia 72h group (DO3 group), 18 rats in each group. Normal control mice breathe room air, and the hyperoxia mice are placed in a closed oxygen chamber (oxygen concentration> 95%). The bronchoalveolar lavage fluid (BALF) cell count and classification, evaluation of lung injury in mice; OPN - / -, OPN + / + mice were exposed to high oxygen exposure time of 20; gelatin zymography analysis of matrix metal The release and activity of MMP2 and MMP9 were detected by RT-PCR. The expressions of OPN, MMP2, MMP9, TIMP1 and TIMP2 mRNA were detected by reverse transcriptase polymerase chain reaction (RT PCR). Results The lung injury in DO3 group was more serious than that in WO3 group. The survival of OPN - / - mice was significantly shorter (χ2 = 23.91, P <0.01). The total number of BALF cells in DO3 group [(72.2 ± 22.3) × 104 / L] was significantly higher than that in WO3 group [(39.7 ± 10.4) × 104 / L, P <0.05] (207.54 ± 36.45) × 103 / L, (25.33 ± 6.43) × 103 / L, P <0.01]. Gelatin zymography showed that BALF-activated MMP9 expression in DO3 group was significantly higher than that in WO3 group [(4.36 ± 0.65) × 104] [(2.84 ± 0.44) × 104, P <0.01]. RT-PCR showed that the expression of OPNmRNA in the lung tissues of WO2 and WO3 groups was significantly higher than that of WN group (0.69 ± 0.04, P <0.05, respectively) (0.87 ± 0.08,0.92 ± 0.07) (0.62 ± 0.09, P <0.05). The expression of TIMP2mRNA in the lung tissues of WO2 and WO3 groups (1.05 ± 0.23 and 0.99 ± 0.13) was significantly different from that in DO and DO3 groups (0.59 ± 0.11, 0.75 ± 0.16, P <0.01, <0.05). Conclusion OPN can inhibit the release and activation of MMP by promoting the expression of TIMP, thereby reducing the ALI induced by hyperoxia.