细胞外基质水凝胶与hiPSC-CM生物相容性的研究  

Biocompatibility of extracellular matrix hydrogel with human iPSCs differentiated cardiomyocytes

作　　者：樊景阳 李苏雷[2] 蒋敏 陶博[2] 曹瑞华[2] 张继彬 田磊[2] 刘建伟[2] 王海滨 曹丰 Fan Jingyang;Li Sulei;Jiang Min;Tao Bo;Cao Ruihua;Zhang Jibin;Tian Lei;Liu Jianwei;Wang Haibin;Cao Feng(Department of Medicine,Qingdao University,Qingdao 266071,China;Second Medical Center,PLA General Hospital,Beijing 100853,China;National Clinical Medical Research Center for Geriatric Diseases,PLA General Hospital,Beijing 100853,China;College of Life Sciences and Bioengineering,Beijing Jiaotong University,Beijing 100044,China)

机构地区：[1]青岛大学医学部,266071 [2]解放军总医院第二医学中心,北京100853 [3]解放军总医院国家老年疾病临床医学研究中心,北京100853 [4]北京交通大学理学院生命科学与生物工程研究院,100044

出　　处：《中华心血管病杂志》2021年第5期487-495,共9页Chinese Journal of Cardiology

基　　金：国家自然科学基金(81820108019);国家重点研发计划(2016YFA0100903,2017YFC1103601)。

摘　　要：目的:观察猪大网膜来源的细胞外基质(ECM)水凝胶与人源诱导多能干细胞分化的心肌细胞(hiPSC-CM)的生物相容性及其作为细胞移植递送载体的可行性。方法:通过化学、物理和酶解的系列方法对猪大网膜组织进行脱细胞处理,然后将提取的ECM制备成可注射性温敏水凝胶,通过组织学染色鉴定其生化成分,用扫描电镜观察其显微表观结构,并向小鼠心肌内注射水凝胶观察其原位凝胶形成能力。采用小分子诱导法将人源诱导性多能干细胞定向分化成心肌细胞,随后将获得的hiPSC-CM分组培养,接种在水凝胶上共培养的为凝胶组,常规培养的为对照组,通过活/死细胞染色、CCK-8和鬼笔环肽染色检测心肌细胞存活状况和生长形态,采用心肌细胞标志物免疫荧光染色及Western blot法分析心肌细胞生存数量和表型维持情况。结果:苏木素-伊红染色、油红O染色和DAPI荧光染色结果显示制备的大网膜ECM水凝胶中无明显的细胞碎片、细胞核和脂质残留,天狼猩红和阿利新蓝染色显示该水凝胶保留了ECM的主要成分胶原蛋白和糖胺聚糖。所制备的水凝胶在4℃条件下表现为黏性液体,在37℃条件下呈凝胶态。扫描电镜结果显示该水凝胶的微观结构由不规则的纤维和大小不一的孔隙组成。在超声引导下可顺利将制备的ECM水凝胶注射到小鼠心肌内,注射后即刻超声下可见高回声信号,提示水凝胶在心肌内滞留,并通过后续的心肌组织苏木素-伊红染色发现注射区有团状凝胶的存在。活/死细胞染色结果显示凝胶组和对照组的hiPSC-CM均大多数存活,很少有死细胞,CCK-8实验结果显示两组吸光度值差异无统计学意义(P>0.05)。鬼笔环肽染色结果显示hiPSC-CM可以在与ECM水凝胶共培养时正常伸展,凝胶组与对照组细胞形态相似,且两组的每细胞F-肌动蛋白覆盖面积差异无统计学意义(P>0.05)。心肌细胞标志物免疫�Objective To observe the biocompatibility of porcine omental derived extracellular matrix(ECM)hydrogel with human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)and the feasibility of ECM hydrogel as a delivery vector of cell transplantation.Methods A series of chemical,physical and enzymatic methods were applied to acellularize the porcine omentum.Subsequently,the extracted ECM was prepared into thermosensitive hydrogel.The biochemical composition of the hydrogel was identified by histological staining.The microstructure was observed by scanning electron microscopy.The hydrogel was then injected into the myocardium of mice to observe its in situ gelation ability.Differentiation of human induced pluripotent stem cells into cardiomyocytes was achieved by small molecule induction,and then the obtained hiPSC-CMs were cultured.hiPSC-CMs cultured onto the prepared hydrogel were defined as the hydrogel group,while conventionally cultured hiPSC-CMs were defined as the control group.Cardiomyocyte viability and growth patterns were detected using live/dead staining,CCK-8 and phalloidin staining.Immunofluorescence staining and Western blot of cardiomyocytes were used to determine the survival and phenotypic maintenance markers of cardiomyocytes in materials.Results The results of HE staining,oil red O staining and DAPI fluorescence staining showed that there was no significant cell debris,nucleus and lipid residue in the prepared ECM hydrogel.The Sirius red staining and Alcian blue staining showed that the hydrogel retained collagen and glycolaminoglycan,which were the main components of ECM.The prepared hydrogel behaves as a viscous liquid at 4℃and as a gel state at 37℃.Scanning electron microscope results showed that the microstructure of the hydrogel was composed of irregular fibers and pores of different sizes.Under the guidance of ultrasound,the prepared ECM hydrogel could be successfully injected into the myocardium of mice.Immediately after the injection,the hyperechoic signal could be observed u

关 键 词：肌细胞 心脏 水凝胶 组织工程 

分 类 号：R318.08[医药卫生—生物医学工程]