【作者】 刘源；

【导师】 张华楸；

【作者基本信息】 华中科技大学 ， 外科学（神经外科方向）（专业学位）， 2024， 硕士

【摘要】 目的:明确CircZXDC/miR-125a-3p/ABCC6调节轴在烟雾病起始因素颈内动脉狭窄并逐渐闭塞中所起作用,并探究其对血管平滑肌具体作用及机制。方法:在既往烟雾病患者外周血RNA测序数据中整理出上调的CircRNA和m RNA、下调的miRNA并通过数据库分析形成调节网络。在收集到的烟雾病患者血浆中检测关键分子CircZXDC,通过ROC曲线检验其对烟雾病早期诊断效果,确立研究关键调节轴为CircZXDC/miR-125a-3p/ABCC6。首先通过双荧光素酶报告基因试验明确CircZXDC与miR-125a-3p,miR-125a-3p与ABCC6的结合情况;应用细胞转染的方法在血管平滑肌细胞中过表达CircZXDC、过表达miR-125a-3p、过表达或敲减下游ABCC6,通过CCK-8细胞活性测定、Ki67细胞免疫荧光染色、Transwell细胞迁移试验、免疫蛋白印记等试验方法,检测血管平滑肌细胞在细胞糖氧剥夺条件下增殖、迁移和转分化情况;使用烟雾病患者颞浅动脉石蜡切片标本进行免疫荧光染色和免疫组化染色检测ABCC6及内质网应激相关蛋白表达;最后在过表达ABCC6的血管平滑肌细胞加入内质网应激抑制剂4-PBA后再次通过CCK-8细胞活性测定、Ki67细胞免疫荧光染色、Transwell细胞迁移试验、免疫蛋白印记等试验检测其增殖迁移能力和转分化情况。结果:CircZXDC在烟雾病患者血浆中高表达,且升高程度与患者是否存在RNF213 p.R4810K基因位点突变有关,并且ROC检测曲线下面积大于0.75。双荧光素酶报告基因试验表明CircZXDC与miR125a-3p,miR125a-3p与ABCC6均存在直接结合。在血管平滑肌中过表达CircZXDC后其CCK-8细胞活性测定、Ki67细胞免疫荧光染色结果表明血管平滑肌的增殖能力提高,Transwell细胞迁移试验与细胞划痕实验示其迁移能力增强,免疫蛋白印记试验表明平滑肌分泌型指标表达上调、ABCC6表达上调,细胞糖氧剥夺条件下可以加剧上述情况。在过表达CircZXDC基础上转染miR125a-3p的模拟物可以逆转上述情况。在烟雾病患者颞浅动脉中ABCC6和内质网应激相关蛋白表达升高,在血管平滑肌啊细胞系中过表达ABCC6后增值迁移能力提高,分泌型指标表达增加,内质网应激相关指标表达增加;而加入内质网应激抑制剂后上述情况得到挽救。结论:本研究验证了烟雾病患者血管平滑肌中存在CircZXDC/miR-125a-3p/ABCC6调节网络,CircZXDC升高导致其吸附的miRNA-125a-3p增多,胞质中剩余和m RNA结合的miRNA-125a-3p减少,ABCC6蛋白翻译增加。而内质网膜上的ABCC6通过增强内质网应激,放大了烟雾病血管缺氧诱导的平滑肌向分泌型的表型转换,导致原本处于中层的血管平滑肌不断增殖,向内膜迁移,且胶原分泌增加,最终导致颈内动脉末端血管狭窄。此外,circ ZXDC可作为一种烟雾病早期诊断的生物标志物;ABCC6特异性抑制剂可以作为烟雾病一种有前景的治疗选择。更多还原

【Abstract】 Objective:To clarify the role of the CircZXDC/miR-125a-3p/ABCC6 regulatory axis in the initiation and gradual occlusion of the internal carotid artery(ICA)in Moyamoya disease(MMD),and explore its specific effects and mechanisms on vascular smooth muscle(VSMC).Methods:Upregulated CircRNAs and mRNA,as well as downregulated miRNAs,were sorted out from peripheral blood RNA sequencing data of patients with Moyamoya disease,and a regulatory network was formed through database analysis.Detect the CircZXDC in the collected plasma of patients with Moyamoya disease,test its early diagnostic effect on Moyamoya disease through ROC curves,and establish the regulatory axis as CircZXDC/miR-125a-3p/ABCC6.Firstly,the binding of CircZXDC with miR-125a-3p and miR-125a-3p with ABCC6 was determined through a dual luciferase reporter gene assay,Using cell transfection methods to overexpress CircZXDC,miR-125a-3p,and overexpress or knock down ABCC6 in VSMC,the proliferation,migration,and transdifferentiation of VSMCs under oxygen glucose deprivation conditions were detected through CCK-8 assay,Ki67 immunofluorescence staining,Transwell assay,and immunoblotting;Immunofluorescence staining and immunohistochemical staining were performed on superficial temporal arteries(STA)to detect the expression of ABCC6 and endoplasmic reticulum stress-related proteins;Finally,after adding endoplasmic reticulum stress(ERS)inhibitor 4-PB A to vascular smooth muscle cells overexpressing ABCC6,their proliferation,migration ability,and transdifferentiation status were detected again through CCK-8 assay,Ki67 immunofluorescence staining,Transwell assay,and immunoprotein blotting.Results:CircZXDC is highly expressed in the plasma of MMD patients,and the degree of elevation is related to the presence of RNF213 p.R4810K mutation in the patient,and the area under the ROC detection curve is bigger than 0.75.The dual luciferase reporter gene assay showed that CircZXDC directly binds to miR125a-3p,and miR125a-3p binds to ABCC6.After overexpression of CircZXDC in vascular smooth muscle,the CCK-8 and Ki67 immunofluorescence staining results showed an increase in the proliferation ability of VSMCs.Transwell test and cell scratch test showed an increase in its migration ability.Immunoprotein blotting test showed an upregulation of smooth muscle secretory markers and ABCC6 expression,which can be exacerbated under OGD.Transfection of miR125a3p mimetics based on overexpression of CircZXDC can reverse the above situation.In MMD STA,the expression of ABCC6 and endoplasmic reticulum stress-related proteins is increased.Overexpression of ABCC6 in VSMCs enhances the ability of migration and secretion,and increases the expression of endoplasmic reticulum stress-related proteins;After adding endoplasmic reticulum stress inhibitors,the above situation was saved.Conclusions:This study confirms the presence of CircZXDC/miR-125a-3p/ABCC6 regulatory network in the vascular smooth muscle of patients with Moyamoya disease.The increase of CircZXDC leads to an increase in the adsorbed miRNA-125a-3p,a decrease in the remaining miRNA-125a-3p bound to mRNA in the cytoplasm,and an increase in the translation of ABCC6 protein.ABCC6 on the endoplasmic reticulum membrane enhances ERS,amplifies VSMCs transdifferentiation which induced by hypoxia in MMD,leading to continuous proliferation and migration of VSMCs to the intima,increased collagen secretion,and ultimately resulting in stenosis of the artery in the end.In addition,circZXDC can serve as a biomarker for early diagnosis of smoke disease;ABCC6 specific inhibitors can be a promising treatment option for MMD.更多还原