摘要
目的研究大鼠急性肺栓塞（APE）后肺组织中线粒体前凋亡蛋白（ARTS）的表达变化及其诱导的细胞凋亡。方法采用自体血栓栓塞颈总动脉制备大鼠APE模型。分别在APE前（对照）和APE后1、8、24和48h开胸取肺脏。常规提取肺组织的总RNA和总蛋白，用半定量逆转录-聚合酶链反应（RT-PCR）方法检测ARTS mRNA表达水平，用蛋白质免疫印迹法（Western blotting）进一步验证ARTS以及ARTS诱导凋亡相关蛋白Bcl-2、Bcl-xL、XIAP和H2Ax的表达变化，用免疫组化法检测肺组织中ARTS在APE前后的表达变化及其组织分布情况，用原位末端缺刻标记法（TUNEL）检测肺组织内细胞凋亡情况。结果APE后ARTS的mRNA及蛋白表达水平均升高，于1h和48h升高最明显（P〈0．05或P〈0．01）。免疫组化显示ARTS在APE前肺组织内表达量很低，不易检测；APE后48h ARTS表达明显升高，主要分布在支气管黏膜上皮细胞和肺泡上皮细胞。TUNEL染色发现，随着ARTS表达升高，肺组织出现明显的细胞凋亡现象，同时ARTS诱导凋亡系统中凋亡抑制蛋白Bcl-2、Bcl-xL和XIAP的表达下降，凋亡标记蛋白H2Ax表达升高（P〈0．05或P〈0．01）。结论大鼠APE后肺组织内ARTS表达明显升高，ARTS介导的凋亡系统在APE后细胞凋亡中发挥了重要作用。
Objective To study the expression changes in apoptosis related protein in transforming growth factors-β signaling pathway （ARTS） in the lung tissues in a rat acute pulmonary embolism （APE） model and its effects on cell apoptosis. Methods A rat APE model was reproduced. Samples of lung tissues were harvested at time points of 1, 8, 24 and 48 hours after APE. Healthy rats were used as control. The changes in mRNA level of ARTS were identified by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR）, and the changes in its protein level, and also Bcl-2, Bcl-xL, XIAP and H2Ax proteins, which were related with ARTS-mediated cell apoptosis, were determined by Western blotting. Immunohistochemical method was employed to study the distribution and expression changes in ARTS in the lung tissue before and after APE. Apoptotic cells in lung tissue sections were identified by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling （TUNEL） method. Results At different time points, the mRNA levels and the protein levels of ARTS significantly increased in the lung tissues of rats with APE at 1 hour and 48 hours （P〈0.05 or P〈0.01）. The immunohistochemical study showed that ARTS had low expression levels and could not be detected in the normal lung tissue, but it was up-regulated obviously at 48 hours after APE, mainly expressed in the bronchial epithelium and the lung alveolar epithelium. Apoptotic cells could be observed in the lung tissue by TUNEL after APE and at the same time when the lung tissue cells exhibited lower levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, and XIAP, as compared with controls. Apoptosis level （as evaluated by H2Ax, apoptotic marker staining） in the lung tissue cells was obviously raised compared with controls （P〈 0.05 or P〈 0.01）. Conclusion The expression of ARTS is up-regulated after APE, and ARTS-mediated apoptosis plays an important role in the cell apoptosis of tung tissue.
出处
《中国危重病急救医学》
CAS
CSCD
北大核心
2008年第6期353-356,I0001,共5页
Chinese Critical Care Medicine
关键词
肺栓塞
急性
蛋白质免疫印迹法
半定量逆转录-聚合酶链反应
细胞凋亡
线粒体凋亡蛋白
acute pulmonary embolism
Western blotting
semi-quantitative reverse transcriptionpolymerase chain reaction
apoptosis
apoptosis related protein in transforming growth factors-β signaling pathway
