【作者】 李楠；

【导师】 李震；

【作者基本信息】 郑州大学 ， 外科学（普外科）（专业学位）， 2019， 硕士

【摘要】 背景胆固醇性脑梗塞和血栓性脑梗塞是缺血性脑梗塞最常见的两种类型。尽管两者在栓子成分、临床表现和治疗上有着明显的区别,但导致两种脑梗塞差异的病理机制并不清楚。目前对胆固醇结晶与缺血性脑梗塞关系的研究也较少。本实验通过建立大鼠脑梗塞模型,旨在对比胆固醇栓塞性脑梗塞与血栓性脑梗塞的差异,并通过全转录组高通量测序探究造成这种差异的潜在分子机制,从而为胆固醇性脑梗塞与血栓性脑梗塞的预防、诊断和治疗提供新的思路。目的构建胆固醇性脑梗塞与血栓性脑梗塞的大鼠模型,对比其梗死体积、神经行为缺陷以及血脑屏障损伤的差异,并通过RNA测序的方式探究两种脑梗塞差异的潜在机制。方法本实验使用微栓子栓塞法构建胆固醇栓塞性脑梗塞(注射胆固醇栓子)和血栓栓塞性脑梗塞(注射血栓栓子)大鼠模型,Longa评分用于评估神经功能缺损,四氯化唑(TTC)染色用于检测梗死体积,依文思蓝(EB)染色用于检测血脑屏障损伤程度。我们进一步使用RNA高通量测序来鉴别两个模型中大脑组织的差异表达的RNA[包括mRNA、长非编码RNA(lncRNAs)、microRNA(mRNAs)和环状RNAs(circRNA)],利用GO和KEGG分析预测差异表达RNA的功能。利用皮尔逊相关系数和MiRanda数据库预测竞争性内源性RNA(ceRNA)对。结果大鼠的脑梗塞模型的实验结果表明,胆固醇组神经功能缺损、梗死体积和血脑屏障损伤程度轻于血栓组,而针对两组的RNA高通量测序发现了1563个mRNA、705个lncRNA、149个circRNA、29个mRNA差异表达,并预测了552个lncRNA-microRNA-mRNA和421个circRNA-microRNA-mRNA的ceRNA关系对。GO和KEGG通路预测了差异表达RNA的潜在功能,其中一些差异表达的RNA广泛参与炎症、细胞生存以及脂质代谢相关的通路,包括MAPK、TNF、IL-17、PI3K、PPAR和L-蛋氨酸合成过程等通路。结论以上结果表明,胆固醇缺血性脑梗塞组脑损伤轻于血栓性脑梗塞组,而RNA高通量测序分析了两组中差异表达RNA,并预测其潜在功能,其中一些mRNA、lncRNA和circRNA可能参与炎症、细胞存活和脂质代谢的相关途径,表明这些差异表达的RNA可能通过这些途径发挥作用,并影响两个脑梗塞大鼠模型的预后。但接下来仍需要进一步的实验来探究这些Scd以及其他的差异RNA参与胆固醇性脑梗塞与血栓性脑梗塞的发病机制,从而为临床的诊断和治疗提供理论依据。更多还原

【Abstract】 BackgroundCholesterol embolic stroke and thrombus embolic stroke are the two most common types of ischemic stroke.Although there are obvious differences in embolic components,clinical manifestations and treatment between the two types of ischemic stroke,the pathological mechanism leading to the difference between the two types of stroke remains unclear.The effect of cholesterol crystals on ischemic stroke is also less concerned.The purpose of this study was to compare the differences between cholesterol embolic stroke and thrombus embolic stroke in rat models,and to explore the underlying pathological mechanism of the differences through transcriptome sequencing,so as to provide new ideas for the clinical prevention and treatment of the two ischemic stroke.ObjectiveTo construct cholesterol embolic stroke model and thrombus embolic stroke model in rats and compare the difference of infarct volume,neurobehavioral deficits and blood-brain-barrier damage,then predict the potential mechanism that leading to the different manifestation between the two stroke model by RNA sequencing.MethodsIn this study,the rat models of cholesterol embolic stroke(by injecting cholesterol emboli)and thrombus embolic stroke(by injecting thrombus emboli)were constructed by the microembolization method.Longa score was used to evaluate neurological deficits,TTC staining was used to detect infarct volume,Evans blue staining was used to detect the degree of blood-brain barrier damage.We further used high-throughput RNA sequencing to identify differentially expressed RNA(including mRNA,long non-coding RNA,microRNA and circular RNA)in brain tissue of the two models,GO and KEGG analysis were used to predict the function of differentially expressed RNA,Pearson correlation coefficient and MiRanda database were used to predict competitive endogenous RNA(ceRNA)pairs.ResultsThe rats model results showed that neurological deficit,infarct volume and blood-brain-barrier damage degree in cholesterol group was lighter than thrombotic group,for RNA-seq,1563 mRNAs,705 lncRNAs,149 circRNAs,29 miRNAs were differentially expressed and 552 ceRNAs of lncRNA-miRNA-mRNA pairs and 421 ceRNAs of circRNA-miRNA-mRNA pairs were predicted.GO and KEGG pathways predict the potential function of differentially expressed RNA.Some differentially expressed RNAs are widely involved in inflammation,cell survival and lipid metabolism related pathways,including MAPK,TNF,IL-17,PI3 K,PPAR pathway and L-methionine synthesis process.ConclusionThese results indicated that brain injury in the cholesterol embolic stroke group was lighter than that in the thrombus embolic stroke group.RNA high-throughput sequencing analyzed the differentially expressed RNAs in the two groups and predicted their potential function.Some of these RNA,lncRNA and circRNA may be involved in the pathways of inflammation,cell survival and lipid metabolism,suggesting that these differentially expressed RNA may play a role through these pathways and affect the prognosis of the two embolic stroke rat models.However,further experiments are needed to explore the mechanism of Scd and other differentially expressed RNAs involved in the pathogenesis of cholesterol embolic stroke and thrombus embolic stroke,so as to provide theoretical basis for clinical diagnosis and treatment.更多还原