胆固醇栓子与血栓栓子所致脑梗塞的对比研究

Cholesterol emboli and blood clot emboli are two major sources that may lead to ischemic stroke found in clinics. However, pathophysiological differences and imaging studies in embolic strokes resulting from these two different emboli have not been well investigated. Based on our preliminary study, we found pathological changes of neurons, includingpyknotic nuclei and eosinophilic cytoplasm, in the hippocampus regions of rats from both cholesterol and blood clot emboli groups. Moreover, more severe damageswere found in cholesterol embolization, compared to blood clot embolization. Though the nature and feature between cholesterol and blood clot emboli are significantly different, the current clinical treatment regimen for embolic strokes is the same internationally. The mechanism and a reliable test method for embolized ischemic stroke need to be investigated. In our study, we used plaques and blood samples which were collected from the patients who underwent carotid endarterectomy, and made them into micro-emboli. We made the ischemic stroke model by injecting the micro-emboli into the rat via internal carotid artery. We will investigate the hemorheology and the damage of the blood brain barrier of the rat’s brain based on molecular biology and PET/CT imaging methods. The purpose of our study is to investigate from the molecular, cellular and histological level to reveal the overall level of the mechanism of the neurological damaged resulted from these two different types of emboli. In addition, we also will perform and analyze the characteristics on PET/CT scan andidentify the similarities and differences between these two types of ischemic strokes. The significance of our study is to reveal the mechanism of cholesterol and blood clot embolized ischemic strokes. It may provide novel treatment strategies in clinics.

胆固醇类栓子和血栓栓子是临床上最常见到的导致缺血性脑梗塞的栓子类型。 然而，这两种栓子所引起的脑梗塞在病理生理及影像学方面的研究少见。我们预实验结果显示胆固醇栓子及血栓栓子所致脑梗塞的大鼠在海马区都发现了神经元细胞固缩及嗜酸性变，而且胆固醇栓子较血栓栓子的损伤更明显。虽然两种栓子在理化性质上有明显不同，但目前国际上对脑梗塞的治疗尚无因此而有具体区别。其作用机制及可靠的检测方法需进一步探讨。本实验是利用病人术后切除的的胆固醇斑块和血液制成的栓子，将其制备成微栓子分别注入大鼠的颈动脉内造成脑梗塞模型。观察大鼠脑血管的血流变学及血脑屏障的损伤变化，利用分子生物学、正电子放射断层造影术以及计算机断层扫描技术（PET-CT）在分子、细胞、组织及动物整体水平探讨脑神经损伤机制并鉴别两种栓子在影像学上的异同。本研究对揭示两种栓子所致脑梗塞的发生机制有重要意义，为临床针对性制定相应的治疗策略提供新的思路。

通过改进的微栓子栓塞法来构建大鼠缺血性脑卒中模型。分别在干预6h、24h对大鼠进行Longa行为学评分，随后取脑皮质区缺血脑组织进行伊文思蓝（Evans Blue，EB）与氯化三苯基四氮唑(2,3,5-Tripheyl Tetrazolium Chloride，TTC)染色，并在术后24h利用串联质量标记法（Tandem Mass Tag,TMT）技术及查库鉴定法检测脑缺血区蛋白表达差异，对差异蛋白进行基因本体(Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析。与血栓栓子组相比，胆固醇栓子组大鼠脑梗死面积较小，而脑组织水肿率及缺血区脑组织EB浓度均无明显差异。蛋白质谱结果显示，胆固醇组与血栓组存在较大的蛋白表达差异，相较于血栓组，胆固醇组中存在12种表达上调的蛋白质（如玻连蛋白等）和3种表达下调的蛋白质（如小凹蛋白）。GO分析提示：差异蛋白主要集中于磷脂代谢过程并发挥与细胞因子受体结合功能。而KEGG通路分析结果表明凝血与补体途径是差异蛋白的主要集中通路。采用基因本体(GO)和京都基因与基因组百科全书(KEGG)通路分析预测差异表达RNA的功能。结果： RNA测序结果显示，脑组织中有1563个mRNA、705个长链非编码RNA (lncRNAs)、149个环状RNA (circRNAs)和29个微型RNA(miRNAs)有表达差异。分别检测出552对lncRNA-miRNA-mRNA 和421对circRNA-miRNA-mRNA的竞争性内源RNA。Go和KEGG基因注解提示差异表达的mRNAs、lncRNAs、circRNAs和miRNAs可能参与胆固醇栓塞性卒中模型的炎症、细胞存活和脂质代谢。在大鼠的缺血性脑卒中模型中，胆固醇栓子组相较于血栓栓子组脑梗死面积更小，并且两组间存在较大的蛋白表达差异。RNA测序结果表明mRNAs、lncRNAs、circRNAs和miRNAs在区分这两种栓塞性卒中类型的分子机制中具有潜在作用。