利用RNA-Seq技术筛选脑血流自动调节相关基因

Investigating Genes Involved in the Changes of Cerebral Autoregulation after Hypertension via RNA-Seq

目的利用RNA-Seq技术筛选并分析与脑血流自动调节相关的基因。方法健康雄性SD大鼠随机分为高血压模型组(14只)和假手术组(6只),高血压模型组大鼠进行双侧肾动脉夹闭,假手术组行开腹手术,不夹闭肾动脉。术后8周将大鼠麻醉,以10～15 mm Hg为一级降低血压,同步记录股动脉血压、颈动脉血流量以及大脑中动脉血流速度,绘制自动调节曲线,并计算脑血流自动调节下限(lower limit of autoregulation,LLCA)。其后取大鼠脑组织进行转录组测序(RNA sequencing,RNA-Seq),比较两组基因表达差异。根据LLCA升高程度将模型组分为两个亚组,高LLCA亚组与低LLCA亚组,高LLCA与假手术组筛选差异表达基因。结果完整采集数据模型组6只,假手术组3只。模型组收缩压、平均动脉压以及LLCA均显著高于假手术组(P<0.05)。模型组中高LLCA亚组与低LLCA亚组收缩压、平均动脉均无明显差异,但高LLCA组的LLCA值高于低LLCA组,差异具有统计学意义(P<0.05)。模型组与假手术组相比有45条基因差异表达,其中上调基因30条,下调基因15条,高LLCA亚组与假手术组比较有478条基因差异表达,其中上调基因289条,下调基因189条。两两比较的交集差异基因共有38条,功能主要涉及趋化作用、平滑肌细胞分化、肌管细胞发育、整联蛋白介导的信号通路等。结论高血压大鼠与正常大鼠共有45条基因差异表达,其中高LLCA的高血压大鼠有478条基因表达发生变化。

Objective This study was designed to investigate the genes involved in the mechanisms underlying cerebral autoregulation via RNA sequencing technique.Methods Total 20 SD rats were randomly divided into hypertension group and sham operation group.In hypertension group,bilateral renal arteries were occluded,and in sham operation group,rats underwent same surgery process except bilateral renal artery occlusion.Eight weeks after surgery,rats were decreased blood pressure gradually by 10 ～ 15 mm Hg per grade,the femoral blood pressure,middle cerebral arterial blood flow velocity and carotid blood flow were monitored substantially,and lower limit of cerebral autoregulation（LLCA） were measured.After that,RNA sequence was performed to screen gene expression of rat brains,and differentially expressed genes between hypertension group and sham operation group were detected.Results Data of 6 rats in hypertension group were collected completely,and 3 in sham operation group.Two subgroups were divided from hypertension group according to data of LLCA,the high LLCA group and less-high LLCA group.In comparison to sham operation group,the systolic pressure（SBP）,mean arterial blood pressure（MABP） and LLCA were significantly higher（P〈0.05） in hypertension group.There were no apparent differences of SBP,MABP and LLCA between high LLCA group and less-high LLCA group.As for the results of RNA-Seq,there are 45 differentially expressed genes between sham operation group and hypertension group,（30 up-regulated by 1.5-fold and 15 down-regulated by 0.75-fold）,478 differentially expressed genes between high LLCA group and sham operation group（289 up-regulated by 1.5-fold and 189 down-regulated by 0.75-fold）.Thirty-eight of these 478 differentially expressed genes were also differentially expressed in hypertension group when compared with sham operation group.The gene ontology（GO） function annotations of the 38 genes mainly involved embryonic organ development,smooth muscle cell differentiation,integrin-mediated signaling pathway,neutrophil chemotaxis,el al.Conclusion There were 45 gene expressions were altered by hypertension,and 478 genes were differentially expressed in high LLCA group when compared with sham operation group.