摘要
目的探索静电纺丝技术制备的有序rGO/PLCL支架对hiPSC-CPC心肌分化的影响。方法采用静电纺丝技术制备有序rGO/PLCL支架。使用扫描电镜观察支架表面结构;通过拉伸试验测定材料机械性能;利用XRD测定支架成分;通过电阻率测定评估支架导电性;种植hiPSC-CPC评估该材料对心肌分化、增殖、形态的影响;通过qPCR、RNA-seq探索rGO对hiPSC-CPC心肌分化基因水平的影响。结果扫描电镜显示,支架材料表面纤维有序排列。拉伸试验显示,在干燥和湿润情况下支架都具有良好机械性能,rGO的加入增加了支架材料的应变能力。XRD验证了rGO/PLCL的成分组成。电阻率测定提示rGO的加入显著增加了支架的导电性。细胞试验显示支架对细胞具有引导作用,rGO的添加使更多的心肌细胞趋于成熟细胞形态,rGO的加入对细胞增殖无明显影响。qPCR结果提示rGO的加入上调了肌节成熟相关基因cTNT、MYH7/MYH6以及细胞间通信相关的连接蛋白CX43的表达。RNA-seq检测提示rGO主要通过下调与细胞外空间、细胞外区域、钙离子结合、细胞外基质组成和排列、细胞黏附等相关基因的表达来影响细胞分化。结论有序rGO/PLCL支架具有有序排列的表面微结构、合适的力学性能以及良好的导电性和生物相容性,能够引导细胞排列,有利于改善hiPSC-CPC分化形成的心肌细胞的形态,以及促进肌节成熟相关基因的表达。
Objective To investigate the effect of aligned rGO/scaffolds prepared by electrospinning on the myocardial differentiation of hiPSC-CPC.Methods Aligned rGO/PLCL scaffolds were prepared by electrospinning.Scanning electron microscope was used to identify the morphology and structure of the scaffolds;Tensile test was used to test the mechanical properties of the scaffolds;XRD was used to determine the composition of the scaffolds;Electrical resistivity was used to evaluate conductivity of scaffolds;Seeding the hiPSC-CPC on the scaffolds to evaluate the effect on cardiomyocyte differentiation,proliferation and morphology;qPCR and RNA-seq were used to explore the effects of rGO on gene level of myocardial differentiation of hiPSC-CPC.Results Scanning electron microscopy showed the scaffolds were aligned.The tensile test showed that the scaffolds had good mechanical properties under dry and wet conditions,and the strain capacity increased with the addition of rGO.The composition of rGO/PLCL was confirmed by XRD.The conductivity of the scaffolds was significantly increased by the addition of rGO.The scaffold can guide cells effectively.And there were more cardiomyocytes tend to be the mature morphology with the addition of rGO.The addition of rGO had no significant effect on cell proliferation.qPCR result suggested that the addition of rGO made the expression of cTNT,MYH7/MYH6 which are related to myosin mature and intercellular communication maker CX43 increased.RNA-seq indicated that rGO affected cell differentiation by down regulating genes related to extracellular space,extracellular region,calcium ion binding,extracellular matrix structural constitution,and cell adhesion.Conclusion The aligned rGO/PLCL scaffolds have aligned surface microstructure,appropriate mechanical properties,excellent conductivity,good biocompatibility which can guide cell arrangement,improve the morphology of cardiomyocytes differentiated from hiPSC-CPC,and promote the expression of myosin maturation related genes.
作者
谭瑶
颜冰倩
艾雪峰
宫艺其
王会景
陈颖
刘明璐
徐徐
王伟
付炜
TAN Yao;YAN Bingqian;AI Xuefeng;GONG Yiqi;WANG Huijing;CHEN Ying;LIU Minglu;XU Xu;WANG Wei;FU Wei(Institute of Pediatric Translational Medicine,Department of Pediatric Cardiothoracic Surgery,Shanghai Children's Medical Center,Shanghai Jiao Tong University School of Medicine,Shanghai 200127,China;Shanghai Key Laboratory of Tissue Engineering,Shanghai 200011,China)
出处
《组织工程与重建外科》
2021年第1期8-18,共11页
Journal of Tissue Engineering and Reconstructive Surgery
基金
国家重点研发计划干细胞及转化研究重点专项(2019YFA0110400)
国家自然科学基金(81670372,82070430)
上海市浦江人才计划(18PJD031)
上海市转化医学协同创新中心(TM201821)
上海市自然科学基金(20ZR1434500)
上海市浦东新区科技发展基金(PKJ2020-Y06)。
关键词
组织工程
静电纺丝
有序支架
还原氧化石墨烯
心肌分化
Tissue engineering
Electrospun
Aligned scaffolds
Reduction of graphene oxide
Myocardial differentiation
