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小鼠心室肌脱细胞化细胞外基质薄片的制备和评价

姜煜东, 李文思, 余翀, 王璐, 孙小夕, 席姣娅^*

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，武汉 430030

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300 μm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, mES-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。mES-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。

关键词： 心肌片; 组织工程; 脱细胞化; 细胞外基质

分类号：R329.3;Q813.1

Acquirement and evaluation of murine ventricular extracellular matrix

JIANG Yu-Dong, LI Wen-Si, YU Chong, WANG Lu, SUN Xiao-Xi, XI Jiao-Ya^*

Department of Physiology and Chinese-German Stem Cell Center, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, the Key Laboratory for Drug Target Researches and Pharmacodynamic Evaluation of Hubei Province, Wuhan 430030, China

Abstract

Cardiac extracellular matrix (ECM), generated from the process of decellularization, has been widely considered as an ideal source of biological scaffolds. However, current ECM preparations are generally difficult to be applied to generate cardiac tissue. Our research was aimed to improve decellularization protocols to prepare cardiac ECM slices. Adult murine ventricular tissues were embedded in low melting agarose and cut into 300 μm slices, and then were divided randomly into three groups: normal cardiac tissue, SDS treated group (0.1% SDS) and SDS+Triton X-100 treated group (0.1% SDS+0.5% Triton X-100). Total RNA content and protein content quantification, HE staining and immunostaining were used to evaluate the removal of cell components and preservation of vital ECM components. Furthermore, murine embryonic stem cell-derived cardiomyocytes (mES-CMs) and mouse embryonic fibroblasts (MEFs) were co-cultured with ECM slices to evaluate biocompatibility. The relative residual RNA and protein contents of ECM slices significantly decreased after decellularization. HE staining showed that SDS+Triton X-100 treatment better destroyed cellular structure and removed nuclei of ECM slices, compared with SDS treatment. Immunostaining showed that collagen IV and laminin were better preserved and presented better similarity to original cardiac tissue in ECM slices acquired by SDS+Triton X-100 treatment. However, collagen IV and laminin were significantly decreased and arranged disorderly in SDS treated group. We observed effective survival (≥ 12 days) of MEFs and mES-CMs on ECM slices acquired by SDS+Triton X-100 treatment, and signs of integration, whereas those signs were not found in SDS treated group. We concluded that, compared with traditional SDS method, new combined protocol (SDS+Triton X-100) generated ECM slices with better component and structural preservation, as well as better biocompatibility.

Key words: myocardium slice; tissue engineering; decellularization; extracellular matrix

收稿日期：2014-05-11　　录用日期：2014-07-09

通讯作者：席姣娅　　E-mail: zhengyall@hotmail.com

引用本文：

姜煜东, 李文思, 余翀, 王璐, 孙小夕, 席姣娅. 小鼠心室肌脱细胞化细胞外基质薄片的制备和评价[J]. 生理学报 2014; 66 (6): 709-717.

JIANG Yu-Dong, LI Wen-Si, YU Chong, WANG Lu, SUN Xiao-Xi, XI Jiao-Ya. Acquirement and evaluation of murine ventricular extracellular matrix. Acta Physiol Sin 2014; 66 (6): 709-717 (in Chinese with English abstract).