【摘要】 目的 通过深度测序技术获得响应脑缺氧、缺血应激的mRNAome与lncRNAome结构变异与基因表达的基础数据，为缺血性脑卒中临床应用研究提供可用的靶标基因以及分子标志物。方法 采用线栓法构建大鼠缺氧、缺血以及再灌注损伤模型，脑切片TTC染色以及行为学评分法评估模型。采用深度测序技术对皮层样本全转录组进行测序，采用生物信息学方法分别鉴定与筛选转录本、转录本结构变异以及差异表达的基因，采用qRT-PCR方法验证差异基因的表达。结果 应用Longa法构建大鼠MCAO模型。缺血缺氧处的脑组织在TTC染色后呈现苍白色，与非梗死区域的红润颜色形成鲜明的对比。高通量测序共产生108.54 G大小的测序数据，分别鉴定出30 829个mRNA和31 183个lncRNA转录本。MCAO组全转录组所产生的SNP、Indel总数显著低于对照Sham组，而mRNA的Indel数目则相对保守，在两组间差异并无统计学意义。mRNA可变剪切体数目显著多于lncRNA,但mRNA和lncRNA转录本的可变剪切体总数目在两组间差异无统计学意义。本研究筛选出2 608个mRNA和551个lncRNA在两组间表达差异存在统计学意义，挑选了9个已被证明响应缺血缺氧而表达上升的已知基因作为双盲测试子，经检索后发现该9个上调基因全部落在2 608个差异表达的基因数据集中，qRT-PCR结果进一步证实该9个基因表达量趋势亦同测序数据表达趋势，说明测序数据较为准确、可靠。结论 一部分影响转录本加工与剪切的mRNA与lncRNA在缺氧缺血以及再灌注损伤后活化，可能抑制了部分转录本的转录以及RNA编辑与加工的能力，进而影响了缺血性脑卒中关键靶基因的表达。更多还原

【Abstract】 Objective To explore the basic characteristics and expression patterns of mRNA transcriptome(mRNAome) and lncRNA transcriptome(lncRNAome) in the cortex tissue of middle cerebral artery occlusion(MCAO) rats by using deep sequencing technology, so as to provide available target genes and molecular markers for ischemic stroke. Methods After the hypoxic-ischemia and reperfusion injury model of MCAO rat model was established with thread embolism method, the availability was estimated with behavioral scoring and TTC staining. The full transcriptome of 10 cortical samples from the MCAO and sham groups were sequenced with deep sequencing technology. The mRNAome, lncRNAome, and their structural variations were identified with bioinformatics methods. The differentially expressed genes(DEGs) were verified with qRT-PCR. Results The MCAO model was successfully established. The brain tissue in the ischemic and hypoxic area showed pale after TTC staining, which was in sharp contrast to the ruddy color of the non-infarcted area. A total of 108.54 G clean sequencing data were obtained from high-throughput sequencing, and 30,829 and 311,183 mRNA and lncRNA transcripts were identified respectively. The total numbers of SNP and Indel produced by the MCAO group were significantly fewer than those by the sham group. However, there was no significant difference in the number of mRNA Indel between the two groups. The number of variants of mRNAome was significantly greater than that of lncRNAome, but there was no significant difference in the total number of variants of transcripts between the two groups. A total of 2,608 differentially expressed mRNA and 551 differentially expressed lncRNA were screened out. Altogether 9 known genes responding to ischemia and hypoxia were identified as the double-blind testers, which were verified in the up-regulated gene dataset in the MCAO group. The qRT-PCR results further confirmed that the expression trends of the 9 genes were the same as the sequencing data, indicating that the sequencing data were reliable. Conclusion A part of mRNA and lncRNA affecting the processing and splicing of transcripts are activated after hypoxic-ischemic and reperfusion injury, which may inhibit the transcription of some transcripts and the ability of RNA editing and processing, and then affect the expression of key target genes in ischemic stroke.更多还原