Let-7靶向抑制BMP2在骨髓间充质干细胞源性外泌体抑制血管钙化中的作用及机制研究

Vascular calcification（VC） is a common clinical problem，and an important risk factors leading to adverse cardio/cerebro-vascular events, but there is no exactly effective prevention or treatment. Our previous study found that the expression of bone morphogenetic protein-2 (BMP2) is closely related to VC, and bioactive substances secreted by bone marrow mesenchymal stem cells (MSCs) could inhibit smooth muscle cells (VSMCs) calcification by downregulating BMP2 expression. However, it is unclear which specific substances works. MicroRNAs are the major component of MSC-derived exosomes (MSC-exosomes), in which let-7 is one of the most abundant microRNAs. And bioinformatics analysis showed that let-7 can targetedly regulate BMP2 expression. But so far, the role of let-7 in VC has not been reported. In the upcoming study，we are going to investigate the effect of MSC-exosomes on BMP2 and VC using in vitro and in vivo assays. Then we will identify the role of let-7 in VC by using let-7 overexpression/knockdown MSC. Furthermore, we will investigate the relevance between let-7 and BMP2 by let-7 overexpression/knockdown VSMC and luciferase reporter assay. Finally，we will identify one or several potential downstream target genes of let-7. Our study will certainly provide new molecular targets and strategies for the prevention or treatment of VC, and provide a new direction for the clinical application of MSC.

血管钙化（VC）是导致心、脑血管恶性事件的重要危险因素，但是目前尚无有效的防治方法。骨形态发生蛋白-2（BMP2）的表达失调与VC密切相关。我们前期研究发现骨髓间充质干细胞（MSC）可以通过分泌某种生物活性物质抑制BMP2表达进而抑制VC。Let-7是MSC源性外泌体中表达量最高的miRNA之一，生物信息学预测其可靶向抑制BMP2表达。迄今为止，let-7在VC中的作用尚未见报道。在此基础上，本项目拟通过细胞及动物实验，证实MSC源性外泌体对BMP2及VC的作用；并建立let-7基因过表达/沉默MSC，证实let-7在VC中的作用；进一步通过基因过表达/沉默、双荧光素酶报告基因检测等技术，精确评价let-7与BMP2的表达相关性；并进一步阐明“let-7～BMP2～下游分子”信号通路。证实MSC源性外泌体通过let-7靶向抑制BMP2抑制VC，为VC的预防和治疗提供新的分子靶点和策略。

动脉粥样硬化疾病（AS）是一种流行于全球的慢性血管炎症性疾病。近年来，以间充质干细胞（MSCs）为基础的治疗方法已被证实在多种疾病具有炎症调控作用，包括AS。然而，MSCs在AS的治疗中疗效不一，受生理性的衰老、培养环境的改变等影响。本研究通过探究正常和多柔比星诱导后MSCs分别在高脂和正常生理环境中分泌的外泌体（exo）对AS进展的作用，进一步探索其中的机制，为提高MSCs治疗疗效提供潜在的思路。以多柔比星诱导MSCs衰老，发现0.25uM多柔比星诱导48小时后，MSCs的表面标志蛋白未发生改变，但是SA-β-Gal染色阳性率高且不出现明显凋亡，衰老保护基因sirt1表达下调，细胞周期相关基因表达改变。Apoe-/-小鼠高脂饮食建立AS模型，隔周注射5×10^5个MSCs，对主动脉行形态学分析以及细胞成分染色。对巨噬细胞的M1和M2亚型进行荧光染色，分析M2/M1占比。发现MSCs能增加斑块内M1及M2型巨噬细胞的数量，Con-MSCs改善巨噬细胞的炎性调控功能和抑制斑块面积，而Dox-MSCs对巨噬细胞的炎性调控功能和斑块的抑制作用下降。通过超速离心的方法提取获得四种不同处理的MSC分泌的exo，获得的exo电镜下呈双层囊泡结构，粒径大小富集在30-200nm。在细胞水平上，将上述四种exo与小鼠巨噬细胞共培养，加以脂质刺激，发现Con-MSCs-exo下调巨噬细胞CD36表达，上调ABCA1和ABCG1的表达，而Dox-MSCs-con对巨噬细胞脂质代谢蛋白的表达调控下降。外泌体进行 miRNAs测序，分析差异性miRNAs，预测靶基因和信号通路。不同组间外泌体miRNAs测序结果显示不同预处理组之间存在差异性miRNAs，且预测的信号通路富集在细胞代谢上。结论：相较于Con-MSCs,Dox-MSCs调控巨噬细胞炎性功能的作用下降和延缓斑块进展的作用减弱。而且，短暂的高脂可增强MSCs对巨噬细胞的炎性调控。这都依赖于MSCs的差异miRNAs表达谱。