血浆miR-125a-5p在川崎病血管内皮损伤及炎症反应中的作用及机制研究

Kawasaki disease is specific genetic background of self-limited autoimmune vasculitis syndrome in children. So far, it lacks the specific early diagnostic markers. Previous, we have confirmed that the plasma miR-125a-5p significantly upregulated in Kawasaki disease (KD). In current study, we will sort the monocytes from the peripheral blood of KD and normal controls and will stimulate with the related inflammation factors, then will collect the exosome from the secretion of the monocytes to detect the expression of miR-125a-5p. The purpose is to determine the original of plasma miR-125a-5p in KD. On the other hand, we will culture THP-1 cell line to express the stable fluorescence miR-125a-5p vector, and collect the secreted exosomes from THP-1, establish the co-culture system between the exosomes and human coronary artery endothelial cells (HCAEC). At same time, we will observe HCAECs proliferation, apoptosis and cytokines secretion by using CCk8, flow cytometry and ELISA methods to prove the plasma miR-125a-5p can transfer into HCAECs by the exosome transportation. Finally, using the luciferase reporter system, WB and real-time PCR, we will confirm that miR-125a-5p can regulate the downstream target genes expression including VEGFA and IL6R to participate in cell proliferation, apoptosis and immune response of vascular endothelial. The purpose of this study is to explain the new mechanisms of the plasma miR-125a-5p upregulation and source in the KD.

川崎病是特定遗传背景下发生的自限性儿童自身免疫性血管炎综合征，缺乏特异性早期诊断标志物。前期我们证实川崎病血浆miR-125a-5p呈特征性高表达，本研究拟通过分离川崎病外周血单核细胞，在相关炎症因子刺激下检测其分泌外泌体中miR-125a-5p表达，明确单核细胞外泌体是其血miR-125a-5p来源。在THP-1细胞稳定转染miR-125a-5p荧光过表达载体，收集外泌体并与人冠状动脉内皮细胞共培养，运用CCK8，流式细胞术和ELISA方法，检测血管内皮细胞增殖、凋亡及炎症因子分泌，证实血浆miR-125a-5p通过外泌体传递至血管内皮细胞并发挥功能。利用荧光素酶报告基因、WB及Real-time PCR方法证实miR-125a-5p通过调控下游靶基因VEGFA和IL6R表达参与血管内皮细胞增殖、凋亡和免疫反应，旨在解答川崎病血浆miR-125a-5p增高的来源及参与川崎病发病的新机制。

川崎病是特定遗传背景下发生的自限性儿童自身免疫性血管炎综合征，缺乏特异性早期诊断标志物。通过本研究，我们证实川崎病外周血单核细胞分泌的外泌体中miR-125a-5p较正常对照组显著高表达，在TNFα炎性细胞因子刺激下，川崎病患儿外周血单核细胞分泌的外泌体中miR-125a-5p变化水平较正常对照显著减低，明确单核细胞外泌体是其血miR-125a-5p来源。通过在THP-1细胞稳定转染miR-125a-5p荧光过表达载体，收集外泌体并与人冠状动脉内皮细胞（HCAEC）共培养，运用CCK8，流式细胞术和ELISA方法，检测HCAEC增殖、凋亡及炎症因子分泌，本研究证实血浆miR-125a-5p通过外泌体传递至血管内皮细胞，导致HCAEC细胞的增殖能力降低、凋亡水平增加及相关炎症细胞因子，包括IL-6分泌水平显著增多。通过本研究我们初步解答了川崎病血浆miR-125a-5p增高的原因及来源的科学问题，同时明确血浆miR-125a-5p能通过外泌体这一传递方式作用血管内皮细胞，从而抑制血管内皮细胞增殖、促进细胞凋亡及调控免疫反应作用的新机制。