GO-AuNPs复合静电纳米纺丝材料促进大鼠BASCs成心肌分化及其调控机制的研究

In recent years, conductive nanomaterials such as graphene (GO) and gold nanoparticles (AuNPs) have been widely used in myocardial engineering. Our preliminary study found that the composite matrix based on carbon nanomaterials can promote the differentiation of brown adipose-derived stem cells (BASCs) into myocardium by enhancing the conductivity of the material, and the AuNPs-based matrix material can promote the development and function mature of cardiomyocytes by changing the local mechanical properties. Recently, we have prepared GO / AuNPs composite electrospinning nanofibers for the first time. Whether this new material can effectively combine the advantages of the two materials and better play the role of promoting BASCs differentiation need to be verified. In this researchr, GO / AuNPs composites were prepared by electrostatic spinning, and their biocompatibility was evaluated. The effects of composite materials on the adhesion, proliferation and differentiation of BASCs were investigated .Its molecular mechanism and key genes and signal pathways are Further explored. This study not only explores the effect of new nanomaterials on BASCs, but also provides a reliable theoretical basis for the application of BASCs and GO / AuNPs composite nanomaterials in myocardial tissue engineering.

近年来导电纳米材料如石墨烯（GO）及金纳米颗粒（AuNPs）在心肌组织工程领域应用日益被关注。我们前期研究发现：基于碳纳米材料的复合基质可通过增强材料导电性促进棕色脂肪干细胞（BASCs）成心肌分化，且基于AuNPs的基质材料可通过改变材料局部力学特性促使心肌细胞闰盘发育和功能成熟。最近，我们首次合成制备了GO/AuNPs复合静电纺丝纳米纤维，这种新型材料能否有效结合两种材料优势，更好地发挥促BASCs分化的作用尚有待验证。本项目拟通过静电纺丝技术制备GO/AuNPs复合材料，并评价其生物相容性；通过系统的形态学及分子生物学方法探究复合材料对BASCs粘附、增殖、分化的作用规律。进一步应用基因表达谱芯片等探究其分子机制，明确关键基因及信号通路。本研究不但可探究新型纳米材料对BASCs的作用规律，还可为基于BASCs及GO/AuNPs复合纳米材料在心肌组织工程领域的应用提供可靠的理论依据。

近年来导电纳米材料如石墨烯（GO）及金纳米颗粒（AuNPs）在心肌组织工程领域应用日益被关注。我们前期研究发现：基于碳纳米材料的复合基质可通过增强材料导电性促进棕色脂肪干细胞（BASCs）成心肌分化，且基于AuNPs的基质材料可通过改变材料局部力学特性促使心肌细胞闰盘发育和功能成熟。本项目利用静电纺丝制备的复合纳米材料薄膜，CCK-8与live-dead结果分析，含0.10mg/ml 的GO的GO/PCL复合材料更适宜细胞的生长，表现出良好的生物相容性，细胞生长状况较其他组好，后续实验以GO浓度0.10mg/ml为实验组。与其他对照组相比，GO/AuNPs复合纳米纺丝材料对棕色脂肪干细胞（BASCs）的增殖分化能力具有明显的促进作用，分化出来的心肌细胞自发收缩能力强，铺展性较好，平均荧光统计结果分析，该复合材料增强了心肌细胞间特异蛋白cTnT与α-actinin以及闰盘相关蛋白connexin-43与NC的表达；Western blotting结果显示：与其他对照组相比，GO/AuNPs组中心肌特异蛋白cTnT与α-actinin以及闰盘相关蛋白CX-43与NC的表达数量明显提高。β1-整合素阻断实验表明β1-整合素抗体同时阻断了缝隙连接蛋白Cx43和力学连接蛋白NC的表达。western blotting检测两组细胞3、7和14d时的p-FAK、Src和ILK的蛋白表达情况，结果表明实验组ILK在各个时间点的表达量均显著高于对照组。通过Western blotting检测两组心肌细胞不同时间点p-AKT等蛋白表达情况，结果表明实验组p-AKT在各个时间点的表达量均显著高于对照组。本研究不但可探究新型纳米材料对BASCs的作用规律，还可为基于BASCs及GO/AuNPs复合纳米材料在心肌组织工程领域的应用提供可靠的理论依据。