放射性脑损伤与缺血缺氧性脑损伤细胞差异分子靶标筛选

Screening of differential molecular targets for radiation brain injury and ischemic-hypoxic brain injury

目的探讨放射性脑损伤(RBI)与缺血缺氧性脑损伤(HIBI)细胞差异分子靶标。方法从GEO数据库中找到相应的数据集后使用GEO2R筛选差异表达基因。并对放射和对照、缺氧缺血及对照之间的通路富集情况做KEGG和GO分析。而后使用PPI分析找出几个关键基因及其蛋白靶标。而后以人星型胶质细胞作为研究对象构建RBI及HIBI模型。在损伤刺激后16 h收集各实验组总细胞和上清中漂浮的坏死细胞,通过RT-PCR和蛋白印迹法分析照射前后以及缺血缺氧处理前后关键基因及其蛋白表达水平之间的差异。结果照射与对照星型胶质细胞共筛选出差异基因237个,其中上调基因95个,下调基因142个。KEGG富集分析结果显示,照射后星型胶质细胞与对照细胞有20条显著差异性富集信号通路(P<0.05)。GO功能分析照射后星型胶质细胞与对照细胞差异显著富集条目62个,其中20个(30.8%)与生物过程(BP)相关,22个(35.4%)与细胞组分(CC)相关,20个(30.8%)与分子功能(MF)相关。缺血缺氧处理与对照星型胶质细胞共筛选出差异基因67个,其中上调基因38个,下调基因29个。KEGG通路富集分析结果显示,缺血缺氧处理后星型胶质细胞与对照细胞有22条显著差异性富集信号通路(P<0.05)。通过差异表达基因的GO功能分析得到显著富集条目35个,其中15个(42.8%)与BP相关,10个(28.6%)与CC相关,10个(28.6%)与分子功能MF相关。STRING分析发现照射后差异表达基因的关键节点基因为EGFR,缺血缺氧处理后差异表达基因的关键节点基因为PHD3。照射组EGFR基因mRNA及蛋白表达水平均显著高于其他两组(P<0.05),缺氧组PHD3基因mRNA及蛋白表达水平均显著高于其他两组(P<0.05)。结论EGFR、PHD3在放射及缺氧缺血处理后的星型胶质细胞中表达有显著差异。

Objective To explore the differential molecular targets of radiation brain injury(RBI)and ischemic-hypoxic brain injury(HIBI).Methods After finding the corresponding data set from the GEO database,GEO2R was used to screen differentially expressed genes.KEGG and GO analysis were performed on the pathway enrichment between radiation group and control group,hypoxia-ischemia group and control group.Then PPI analysis was used to find several key genes and their protein targets.Then,human astrocytes were used as the research objects to construct RBI and HIBI models.The total cells and the floating necrotic cells in the supernatant of each experimental group were collected 16 hours after the injury,and the differences between the key genes and their protein expression levels before and after irradiation and before and after ischemia and hypoxia treatment were analyzed by RT-PCR and Western blotting.Results A total of 237 differential genes were screened between astrocytes after irradiation and control astrocytes,including 95 up-regulated genes and 142 down-regulated genes.The results of KEGG enrichment analysis showed that there were 20 significantly differentially enriched signaling pathways between astrocytes after irradiation and control astrocytes(P<0.05).GO functional analysis showed that 62 entries were significantly enriched between astrocytes after irradiation and control cells.Then,20(30.8%)were related to biological process(BP),22(35.4%)were related to cellular component(CC),and 20(30.8%)were related to molecular function(MF).A total of 67 differential genes were screened between astrocytes treated with astrocytes after ischemia and hypoxia and control astrocytes,including 38 up-regulated genes and 29 down-regulated genes.The results of KEGG pathway enrichment analysis showed that there were 22 significantly different enrichment signal pathways between astrocytes after ischemia and hypoxia and control astrocytes(P<0.05).Through the GO function analysis of differentially expressed genes,35 significantly enriched items were obtained,of which 15(42.8%)related to BP,10(28.6%)related to CC,and 10(28.6%)related to molecular function MF.STRING analysis found that the key node genes of differentially expressed genes after irradiation were EGFR,and the key node genes of differentially expressed genes in astrocytes after ischemia and hypoxia were PHD3.The expression levels of EGFR gene mRNA and protein in the irradiation group were significantly higher than those in the other two groups(P<0.05).The mRNA and protein expression levels of PHD3 genes in the hypoxia group were significantly higher than those of the other two groups(P<0.05).Conclusion There are significant differences in the expression of EGFR,PHD3 in astrocytes after radiation and hypoxic-ischemic treatment.