小鼠心室肌脱细胞化细胞外基质薄片的制备和评价

发布时间：2019-04-01 09:53

【摘要】：心肌细胞外基质(extracellular matrix,ECM)可由心肌经脱细胞处理制得,被广泛认为是一种理想的制备工程心肌的生物支架材料。然而目前的脱细胞方法尚存在不足,本研究拟联合使用经典去垢剂改良脱细胞方法,制备性能更为优良的心肌ECM薄片,以用于构建工程心肌片。用振荡切片机将包埋于低熔点琼脂糖中的成年昆明小白鼠心室肌组织沿心脏横轴切成300μm厚的薄片,随机分为正常对照组、SDS脱细胞组(0.1%SDS处理)和改良脱细胞组(0.1%SDS和0.5%Triton X-100联合处理)。通过总RNA和总蛋白质含量分析、HE染色和免疫荧光染色等方法评估各组的脱细胞程度和ECM成分保留状态;将改良脱细胞组ECM与小鼠胚胎干细胞源心肌细胞(murine embryonic stem cell-derived cardiomyocytes,m ES-CMs)和小鼠胚胎成纤维细胞(murine embryonic fibroblasts,MEFs)共培养以检测其生物相容性。结果显示:SDS脱细胞组和改良脱细胞组ECM中残留的总RNA及蛋白质含量均低于对照组。HE染色结果显示改良脱细胞组核质去除较SDS脱细胞组更彻底。改良脱细胞组可见胶原蛋白IV和层粘连蛋白两种ECM关键成分表达量和分布接近正常心肌组织,而SDS脱细胞组中这两种蛋白明显减少且分布紊乱。m ES-CMs和MEFs能存活于改良脱细胞组ECM表面12天以上并向内迁移。综上,SDS和Triton X-100联合脱细胞法制备ECM薄片效果明显,能更好地保留天然ECM成分和结构,具有良好的生物相容性。
[Abstract]:Myocardial extracellular matrix (extracellular matrix,ECM), which can be prepared by acellular treatment of myocardium, is widely regarded as an ideal biomaterial for preparing engineered myocardium. However, there are still some shortcomings in the present acellular method. In this study, we intend to use the classical scale remover to improve the acellular method to prepare the better performance of myocardial ECM thin slices for the construction of engineering myocardial slices. The ventricular muscle tissue of adult Kunming mice embedded in agarose at low melting point was cut into 300 渭 m thick slices along the transverse axis of heart by oscillatory slicing machine and randomly divided into normal control group. SDS acellular group (0.1%SDS treatment) and modified acellular group (0.1%SDS and 0.5%Triton Xa 100 combined treatment). Total RNA and total protein content analysis, HE staining and immunofluorescence staining were used to evaluate the acellular degree and the retention status of ECM components in each group. The modified acellular group ECM was co-cultured with mouse embryonic stem cell-derived cardiomyocytes (murine embryonic stem cell-derived cardiomyocytes,m ES-CMs) and mouse embryonic fibroblasts (murine embryonic fibroblasts,MEFs) to detect their biocompatibility. The results showed that the contents of total RNA and protein in ECM of SDS acellular group and modified acellular group were lower than those of control group. The results of HE staining showed that the nucleoplasmic removal of modified acellular group was more complete than that of SDS acellular group. In the modified acellular group, the expression and distribution of collagenin IV and laminin ECM were close to normal myocardial tissue. M-ES-CMs and MEFs could survive more than 12 days on the surface of ECM in the modified acellular group and migrate inward. In the SDS acellular group, the expression of these two proteins was significantly decreased and the distribution of the two proteins was disordered. In conclusion, the preparation of ECM slices by acellular method of SDS and Triton X + 100 has obvious effect, which can retain the natural ECM composition and structure better, and has good biocompatibility.
【作者单位】：华中科技大学同济医学院基础医学院生理学系中德干细胞中心湖北省药物靶向研究评价重点实验室;
【基金】：supported by the National Natural Science Foundation of China(No.31100828) the Natural Science Foundation of Hubei Province,China(No.2011CDB363) the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry,China,the Fundamental Research Funds(No.2013TS145) the Fundamental Research Funds for the Undergraduates(No.HUST:2011B142,2012B366)in Huazhong University of Science and Technology the National Undergraduate Training Programs for Innovation and Entrepreneurship(No.HUST:2012175) the Science and Technology Innovation Funds for the University or College Students,China(No.9)
【分类号】：R318.08

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