棉鼠肺表面活性相关蛋白A基因生物信息学分析及其与肺损伤相关性

Bioinformatics analysis of surfactant-associated protein A in cotton rat and its relationship with lung injury

目的 分析棉鼠肺表面活性相关蛋白A（SP-A）的基因序列结构和生物信息学特点,观察棉鼠肺损伤模型中SP-A mRNA和蛋白的表达水平,初探SP-A表达规律与肺损伤的相关性。方法 将32只棉鼠随机均分为4组：3个实验组棉鼠分别腹腔注射2mg/kg脂多糖（LPS）处理24、48和96h,对照组腹腔注射等量生理盐水。建模后提取棉鼠肺组织总RNA,经RT-PCR扩增SP-A基因,并对其进行生物信息学分析;组织切片观察LPS作用不同时期肺组织病理学变化;qRT-PCR分析SP-A mRNA的表达水平;蛋白质印迹法检测SP-A蛋白表达。结果 棉鼠SP-A基因编码区长744bp,编码248个氨基酸,具有多个半胱氨酸保守位点、α螺旋结构和糖基化位点,与其他物种相比其核苷酸（75.4%-90.1%）和氨基酸（70.6%-87.1%）序列均具有较高的同源性。在LPS诱导肺损伤模型中发现,与对照组相比,实验组棉鼠肺组织病理学改变随LPS刺激时间延长而加重,具有时间依赖性;SP-A mRNA和蛋白的表达水平在LPS处理24h后开始迅速增加,差异有统计学意义（P〈0.001和P〈0.01）,48h时仍持续上升（P〈0.001和P〈0.01）,96h时略有下降,但仍保持在较高水平,与对照组相比差异有统计学意义（P〈0.05和P〈0.01）。结论 棉鼠SP-A基因具有高度保守性;棉鼠SP-A mRNA和蛋白的表达水平与肺损伤严重程度密切相关,可反应肺损伤的不同时程。

Objective To analyze the pulmonary surfactant-associated protein A （SP-A） gene sequence and its bioinformatic characteristics and to observe SP-A mRNA and protein levels in lung injury model of cotton rats, so as to explore the correlation between lung injury and SP-A expression . Methods A total of 32 cotton rats were randomly divided into control （normal saline,NS） group and three lipopolysaccharide （LPS）-induced lung injury groups （the cotton rats were injected intraperitoneally with 2 mg/kg LPS for 24,48 and 96 hours,respectively）. The total RNA was extracted from lung tissue and SP-A gene was amplified by RT-PCR. Then bioinformatic analysis was done for SP-A characteristics. Histopathological changes of lung tissue were observed at different time points by hematoxylin-eosin staining; the mRNA level of SP-A was detected by real time-PCR and the protein level of SP-A was detected by Western blotting analysis. Results The coding region of cotton rat SP-A gene had 744 bp and encoded 248 amino acids, containing six cysteine conserved sites, four a-helices and two predicted N-glycosylation sites. Meanwhile,cotton rat SP-A shared high level of homology in the nucleotide sequences （75. 4%- 90. 1%） and in deduced amino acid sequences （70. 6%-87. 1%） with other species. Moreover, histopathological changes of lung tissues in three LPS groups were more notable and severe than those in the control group, and the changes were related to LPS treatment time. Compared with the control group,the mRNA and protein levels of SP-A in lung tissues were significantly increased in three LPS groups, with rapid increase starting from 24 h after treatment （P〈0 . 001 , P〈0 . 01） , with continuous increase at 48 h （P〈0. 001 and P〈0. 0 1 ） ,and slight decrease and still keeping at a high level at 96 h （P〈0. 05, P〈0. 01）. Conclusion SP-A gene of cotton rat is highly conservative; the mRNA and protein levels of SP-A are closely associated with the severity of lung injury.