大鼠急性肺栓塞后SMP-30表达下降及其对细胞凋亡的影响

目的：研究大鼠急性肺栓塞模型肺组织中衰老标记蛋白质30(SMP-30)的表达变化及其对Fas诱导的细胞凋亡的影响。方法:建立大鼠急性肺栓塞模型，分别在急性肺栓塞后1、8、24和48 h进行支气管肺泡灌洗，然后开胸取出肺组织。常规提取肺组织的总RNA和总蛋白，以正常组为对照，采取半定量RT-PCR的方法研究SMP-30在mRNA水平表达的变化；采用Western blotting方法进一步验证SMP-30在蛋白水平表达的变化；采用免疫组织化学方法检测大鼠肺组织中SMP-30以及肺泡巨噬细胞中IL-8在肺栓塞前后表达的变化及其组织分布情况；采用TUNEL法研究急性肺栓塞后组织细胞的凋亡情况；最后采用ELISA法检测急性肺栓塞后肺泡灌洗液中sFasL的浓度变化。结果:在大鼠急性肺栓塞后的不同时点，SMP-30的mRNA水平和蛋白水平均逐渐降低，在24和48 h下降最为明显。免疫组化研究表明SMP-30主要分布在支气管黏膜上皮细胞和肺泡上皮细胞，急性肺栓塞后SMP-30在上述细胞内的表达均明显降低。TUNEL染色发现随着SMP-30表达的降低，肺组织内出现明显的细胞凋亡现象，同时肺泡灌洗液中sFasL的浓度升高，肺泡巨噬细胞内IL-8的表达也明显升高。结论:大鼠急性肺栓塞后肺组织内SMP-30的表达明显降低，可能促进Fas－FasL细胞凋亡系统的活化。

Down-regulation of SMP-30 and its effects on the Fas-mediated apoptosis in the lung tissues after acute PE

AIM: To study the expression changes of senescence marker protein-30 (SMP-30) in the lung tissues of rat acute pulmonary embolism (PE) model and its effects on the Fas-mediated apoptosis. METHODS: A rat acute PE model was constructed. The samples of bronchoalveolar lavage fluid (BALF) and lung tissues were collected successively at different time points of 1 h, 8 h, 24 h and 48 h.The normal rats were used as control. The mRNA level changes of SMP-30 were identified by semi-quantitative RT-PCR. The changes of SMP-30 protein were validated by Western blotting. The immunohistochemical method was employed to study the distribution and expression changes of SMP-30 in the lung tissues and the expression of IL-8 in alveolar macrophages after PE. The apoptotic cells in lung tissue section were identified by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method. BALF sFasL levels were measured with an enzyme-linked immunosorbent assay (ELISA) system. RESULTS: At different time points, the mRNA levels and the protein levels of SMP-30 decreased in the lung tissues of rat acute PE models, obviously at 24 and 48 h. The immunohistochemical study indicated that SMP-30 was mainly expressed in the bronchial epithelium and the lung alveolar epithelium, and was down-regulated obviously after PE. The apoptotic cells were observed in the lung tissues after acute PE and at the same time the BALF concentrations of sFasL and the expression of IL-8 in alveolar macrophages increased obviously compared with controls. CONCLUSION: The expression of SMP-30 is down-regulated after acute PE, indicating some relationships with the activation of Fas-FasL system in the lung tissues.