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目的探索制备离体大鼠心脏脱细胞生物支架材料的新方法,为心脏组织工程研究提供三维立体天然支架。方法取30只成年SD大鼠心脏,运用冻融加化学萃取的组织工程学方法(胰蛋白酶、十二烷基硫酸钠和曲拉通X-100)处理离体大鼠心脏,同时观察心脏大体形态及颜色变化,并对脱细胞支架进行基因组DNA分析;HE染色,免疫荧光法,扫描和透射电镜进一步检测鉴定脱细胞支架的生物学特征。结果心脏脱细胞支架外观透明,包膜完整,维持心脏三维立体结构,肉眼可见心脏内脉管系统;脱细胞支架DNA残留量不及对照组的3%;HE染色、扫描和透射电镜结果显示,心脏脱细胞生物支架去细胞彻底,细胞外基质网状结构保留完整;免疫荧光结果表明,胶原、弹性蛋白等细胞外支架成分保留较完整,未见明显细胞核成分残留。结论运用冻融加化学萃取法所制备的离体心脏脱细胞生物支架去细胞彻底,细胞外基质保留较完整,是较为理想的心脏三维立体生物支架材料。
Objective To explore a new method for preparing cardiac acellular biological scaffolds in vitro and to provide a three-dimensional natural scaffold for cardiac tissue engineering. Methods The hearts of 30 adult SD rats were obtained and the isolated rat hearts were treated by freeze-thaw and chemical extraction of tissue engineering (trypsin, sodium lauryl sulfate and Triton X-100) Morphological and color changes, and the acellular scaffold for genomic DNA analysis; HE staining, immunofluorescence, scanning and transmission electron microscopy to further identify the biological characteristics of acellular scaffold. Results The appearance of acellular acellular scaffolds was clear and the envelope was intact. The three-dimensional structure of the heart was maintained and the intravascular system of the heart was visible. The residual DNA of the acellular scaffold was less than 3% of the control group. HE staining, scanning and TEM showed that the heart The acellular biological scaffolds were completely decellularized and the extracellular matrix reticular structure remained intact. The results of immunofluorescence showed that the components of extracellular scaffolds, such as collagen and elastin, remained relatively intact and no obvious nuclear components remained. Conclusion The isolated cardiac acellular biological scaffolds prepared by freeze-thaw and chemical extraction method are completely decellularized and the extracellular matrix remains relatively intact, which is an ideal cardiac three-dimensional bio-scaffold material.