放射性肺损伤小鼠晚期转录水平特征性基因标志物的研究
Transcriptional investigation of biomarkers for late radiation-induced lung toxicity in mice
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摘要:目的 研究不同剂量X射线照射小鼠肺部后的差异基因并进行转录水平分析，探讨小鼠放射性肺损伤（RILI）潜在的基因标志物。方法 8周龄C57BL6雌性小鼠96只，X射线单次照射分为3组（12只/组）：对照组（未接受照射）、中等剂量组（MD组，10 Gy单次照射）、高剂量组（HD组，20 Gy单次照射），余60只分为5组进行X射线全肺野梯度剂量照射。采用全基因组表达芯片对小鼠肺组织进行RNA检测并生成基因表达矩阵，使用R语言软件包进行基因表达数据转换，对特征性基因标志物表达水平进行验证与数学建模，对HD组小鼠基因表达水平进行基因本体论生物学功能基因集富集分析。采用经典贝叶斯检验方法进行组间基因表达差异的比较，采用线性回归模型分析2组独立数据的关联程度（关联系数为R2）。结果 MD组和HD组小鼠照射后肺组织转录组学分析，共筛选出RILI差异表达基因539个，其中差异最显著的5个基因为Phlda3、Fgg、Kng1（上调基因）和Ptprb、Kit（下调基因）。梯度剂量X射线分次照射实验结果证实，3个上调基因均随X线剂量的增加而表达增强；2个下调基因则随剂量的上升而表达降低。上调基因的逻辑回归模型拟合程度较高（χ2=11.66, R2=0.88）；下调基因的表达值与剂量具有良好的相关性（R=−0.95，R2=0.89）。基因集富集分析结果显示，上调基因富集于p53信号通路、固有免疫反应等生物学过程，下调基因富集于细胞代谢、肺部发育等生物学过程。结论 上调基因Phlda3、Fgg、Kng1与下调基因Ptprb、Kit可作为客观反应RILI严重程度的潜在基因标志物，为日后开展临床与辐射防护相关应用奠定前临床基础。Abstract:Objective To investigate differentially regulated genes after whole thoracic X-ray exposure and to explore the potential gene markers at transcriptome levels of radiation-induced lung injury (RILI) at transcriptome levels in mice.Methods A total of 96 C57BL6 female mice aged 8 weeks were divided into three groups by single X-ray irradiation, namely. control group (no irradiation), medium dose group (MD group, 10 Gy single irradiation), and high dose group (HD group, 20 Gy single irradiation). Whole thoracic X-ray irradiation was delivered to the remaining 60 mice with a wide range of doses and fractionations. Whole genome expression chips were used in the detection of RNA in mouse lung tissues and gene expression data were converted by the R language software. Classical Bayesian test was used in exploring differentially expressed genes between groups. The expression levels of key genes were validated and mathematically analyzed, and gene ontology biological function enrichment analysis was performed. The correlation degree between two groups with independent data was analyzed by regression model (correlation coefficient is R2).Results According to medium vs. high dose irradiations, the differentially regulated genes(539 genes) were selected. Then, the top five most significant genes were identified, namely, Phlda3, Fgg, Kng1 (up-regulated genes) and Ptprb, Kit (down-regulated genes). Dose escalation studies confirmed that the transcriptional status of the five gene signatures correlated well with radiation doses. The expression levels of the three up-regulated genes increased with the boost of X-ray dose (logistic regression model χ2=11.66, R2=0.88); whereas the expression levels of the two down-regulated genes decreased with the boost in X-ray dose (linear regression R=−0.95, R2=0.89). Gene set enrichment analysis revealed that up-regulated genes were associated with p53 signaling and innate immune response; whereas the down-regulated genes were enriched in biological processes, such as cell metabolism and lung development.Conclusions Up-regulated genes Phlda3, Fgg, Kng1, as well as the down-regulated genes, Ptprb and Kit, can be used as potential genetic markers to indicate the severity of RILI. This finding sheds light on the mechanism involved in the radiation protection evidenced by mRNA biomarkers.
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