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目的研究高温相关丝氨酸蛋白酶A2(Omi/HtrA2)特异性抑制剂(Ucf-101)对早产鼠高体积分数氧(高氧)肺损伤的保护作用。方法 72只新生早产Wistar大鼠在出生12 h随机分为对照组、高氧组、高氧+Ucf-101组,每组24只。高氧+Ucf-101组新生大鼠高氧暴露前10 min,按1.5μmol·kg-1腹腔注射Ucf-101,对照组和高氧组新生大鼠在相同时点注射等量的9 g·L-1盐水;高氧组和高氧+Ucf-101组新生大鼠持续暴露于950 mL·L-1氧气中,对照组新生大鼠置于同一室内常压空气中。分别于暴露1 d、3 d、7 d时,每组取动物8只,留取肺组织标本。左肺制作石蜡切片,采用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶连结法检测Omi/HtrA2、X连锁凋亡抑制蛋白(XIAP)和半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)在各组肺组织的表达;采用原位末端标记法检测肺细胞凋亡情况。右肺制作冷冻切片,采用间接免疫荧光双染色法,在激光共聚焦显微镜下观察Omi/HtrA2在细胞内的转位。结果 1.高氧组和高氧+Ucf-101组肺组织Omi/HtrA2、Caspase-9随着高氧暴露时间延长表达量呈增高趋势,与对照组比较差异有统计学意义(Pa<0.05);高氧+Ucf-101组与高氧组比较,Omi/HtrA2、Caspase-9在同一时间点表达量明显降低,差异均有统计学意义(Pa<0.01)。高氧组和高氧+Ucf-101组XIAP较对照组明显减少,差异有统计学意义(P<0.01);高氧+Ucf-101组各时间点XIAP的表达量均显著高于高氧组,差异有统计学意义(P<0.01)。2.与对照组比较,高氧组和高氧+Ucf-101组肺组织细胞凋亡率明显增加(P<0.01);高氧+Ucf-101组与高氧组比较,同一时间点凋亡率显著减低(P<0.01)。3.高氧组和高氧+Ucf-101组肺组织Omi/HtrA2胞内转位率明显高于对照组(P<0.01),高氧+Ucf-101组肺组织Omi/HtrA2转位率较同一时间点的高氧组明显降低,差异有统计学意义(P<0.01)。结论 Ucf-101可能通过减少Omi/HtrA2在肺细胞胞质中的表达和线粒体的转位而抑制肺细胞凋亡,减轻早产鼠高氧肺损伤。
Objective To investigate the protective effects of high temperature-associated Omi / HtrA2-specific inhibitor (Ucf-101) on high volume fraction oxygen (hyperoxia) lung injury in premature rats. Methods Seventy two neonatal Wistar rats were randomly divided into control group, hyperoxia group and hyperoxia + Ucf-101 group, with 24 rats in each group. The neonatal rats in hyperoxia + Ucf-101 group were given intraperitoneal injection of Ucf-101 10 min before hyperoxia exposure, and the control and hyperoxia groups were injected with the same amount of 9 g · L-1 saline. The neonatal rats in hyperoxia group and hyperoxia + Ucf-101 group were exposed to 950 mL · L-1 oxygen continuously. The neonatal rats in the control group were placed in the same room of atmospheric air. At 1 d, 3 d and 7 d after exposure, 8 animals in each group were taken for lung tissue samples. Paraffin sections were made in the left lung and the expression of Omi / HtrA2, X-linked inhibitor of apoptosis protein (XIAP) and caspase-9 Caspase-9) expression in lung tissue of each group; lung cell apoptosis was detected by in situ end labeling. Frozen sections were made in the right lung. Indirect immunofluorescence double staining was used to observe the translocation of Omi / HtrA2 in the cells under confocal laser scanning microscope. The results showed that the expression of Omi / HtrA2 and Caspase-9 in lung tissues of hyperoxia group and hyperoxia + Ucf-101 group increased with the prolongation of hyperoxia exposure, and the difference was statistically significant (P <0.05) Compared with hyperoxia group, the expression of Omi / HtrA2 and Caspase-9 in hyperoxia + Ucf-101 group was significantly decreased at the same time point, the difference was statistically significant (Pa <0.01). Compared with the control group, the levels of XIAP in hyperoxia + Ucf-101 group were significantly lower than those in the control group (P <0.01), and those in the hyperoxia + Ucf-101 group were significantly higher than those in the hyperoxia group , The difference was statistically significant (P <0.01). Compared with the control group, the apoptosis rate of lung tissue in hyperoxia group and hyperoxia + Ucf-101 group increased significantly (P <0.01); Compared with hyperoxia group, the apoptosis rate in the hyperoxia + Ucf-101 group at the same time The rate was significantly lower (P <0.01). The intracellular transposition rate of Omi / HtrA2 in hyperoxia group and hyperoxia + Ucf-101 group was significantly higher than that in control group (P <0.01). The translocation rate of Omi / HtrA2 in lung tissue in hyperoxia + Ucf-101 group was Hypoxia group at the same time point was significantly lower, the difference was statistically significant (P <0.01). Conclusion Ucf-101 may inhibit lung cell apoptosis and reduce hyperoxia-induced lung injury in premature rats by decreasing the expression of Omi / HtrA2 in the cytoplasm of lung cells and the translocation of mitochondria.