基于唐氏模型小鼠Tc1探讨可防治动脉粥样硬化的miRNA子集及其靶向输送策略

Atherosclerosis (AS) is a life-threaten disease, due to the possible eruption of the vulnerable plaques. Elucidation of the underlying mechanism and development of the therapeutic strategies is of great theoretical and clinical importance. Accumulating clinical evidence indicates that patients with Down syndrome, who harbor one additional copy of Chromosome 21, have much lower incidence of atherosclerosis, suggesting that certain genes in the Chromosome 21 should be anti-atherogenic. Identification and characterization of these protective factors are pre-requisite for therapeutic delivery to the diseased blood vessels, both of which would shed light on the treatment of atherosclerosis. With the support of the NSFC (81170149), we have imported the Tc1 mice, which harbor the most portion of the human chromosome 21 and mimic the phenotype of Down syndrome. We have revealed that Tc1 mice display lower impairment of the endothelium and infiltration of immune cells under high fat diet, and plaques were rarely found. There are estimated 232 genes in the transferred Chromosome 21, with miRNAs included. Functional cluster and other bioinformatics analysis imply that miR-99/125/let7c are possibly the potent anti-atherogenic factors. The proposed project would first confirm the protective role of the miRNAs candidates miR-99/125/let7c in vitro and in vivo, by combinatorial use of control and Tc1 mice. The cellular and molecular mechanisms for their protective roles would be also clarified. With the advance of exosome in targeted drug delivery, we next would focus on deliver the above protective miRNAs to the diseased blood vessels by modification of the natural exosome. Briefly, Lamp2b protein, the key components of exosome membrane, is fused with RGD by molecular cloning, which would specifically recognize the inflammatory endothelium. After purification of the exosome, candidate miRNAs drugs would be loaded into the exosome by electroporation. The targeted specificity and therapeutic efficacy would be systemically evaluated by both histology and echocardiography. The current proposal would be promisingly find novel anti-atherogenic genes and establish new treatments for this disease.

动脉粥样硬化（AS）严重威胁人类生命健康，探讨其发病机制及干预策略具有重要意义。临床研究表明，唐氏综合征（又称21-三体综合征）患者AS发病率低，提示21号染色体上存在重要的AS保护因子。我们前期引进唐氏模型小鼠Tc1（NSFC 81170149），发现该模型小鼠高脂饮食后几乎无斑块发生；对其中额外的232个基因进行深入的功能聚类和生物信息学分析发现，miR-99/125b/let7c可调控多条AS相关通路，提示这些miRNAs可能是重要的AS保护因子。本项目拟在细胞和整体动物水平上，明确上述miRNAs在抑制AS发生中的作用和具体机制；此外，结合外泌体药物靶向输送优势，设计靶向受损血管的外泌体并加载上述miRNAs，系统评价改构外泌体对小鼠AS的靶向性及对AS发生发展的影响。本项目有望发现新的AS保护因子，创建一种新的AS靶向输送基因药物方法，为动脉粥样硬化的防治提供全新的靶向干预策略。

动脉粥样硬化（AS）严重威胁人类生命健康，探讨其发病机制及干预策略具有重要意义。本项目通过唐氏模型小鼠筛选AS密切相关分子，并创建了基于外泌体的AS靶向输送基因药物方法，为动脉粥样硬化的防治提供全新的靶向干预策略，取得了以下几方面的结果：1）我们发现了一组miRNA（miR33，miR125b，let-7，miR155）与AS发生发展密切相关。2) 基于生物信息分析筛选候选miRNA中的miR125b作为研究对象，并在AS小鼠验证了miR-125b是动脉粥样硬化的致病因子。 3）我们基于外泌体创建了新的抑制巨噬细胞miR155表达的策略，具有理想的改造M1、M2平衡的效果，该策略为后续干预巨噬细胞极化提供了新的备选方案。4) 基于高通量测序分析血压正常志愿者与高血压初诊患者血浆外泌体中的差异miRNA，筛选差异显著的miR-320作为研究对象，并初步验证exo-miR-320可作为判断血管内皮损伤的生物标志物，及其对血管损伤及动脉粥样硬化斑块影响。.此外，在本项目的支持下，课题组开辟了“超声联合外泌体在心血管疾病诊疗中的应用”这一个重要研究方向，主要围绕动脉粥样硬化（AS）和化疗引起的心脏损伤等心血管疾病的新策略方面进行系列研究，展开药物装载、药物递送、疾病应用三个部分的研究，取得了阶段性的进展，主要包括：1）在药物装载方面，我们利用RNA结合蛋白创建外泌体高效装载目的RNA新策略；2）在药物递送方面，建立了基于超声靶向爆破技术（UTMD），提高外泌体靶向递送效率的新策略；2）在疾病应用方面：①发现肝脏靶向递送LDL-R改善缺陷型小鼠AS情况，有望在临床上用于家族性高胆固醇血症患者的治疗；②发现棕色脂肪组织外泌体（BAT-Exos）能够缓解肥胖小鼠的代谢综合征；③利用UTMD辅助外泌体递送策略，探讨递送miR-21保护化疗心脏损伤。

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