一种改良的去细胞皮瓣制备方法

Optimized preparation method for decellularized skin flap

目的:探索一种简易的去细胞皮瓣制备方法,并对制备完成的去细胞皮瓣的生物学特征进行分析。方法:取成年大鼠下腹壁处皮瓣去血后,使用磁力搅拌器进行辅助震荡法去细胞。对所获支架通过DNA定量分析、SDS残留量检测、扫描电镜等检测获取其生物学特征数据。再取皮下包埋于成年SD大鼠背部的去细胞皮瓣标本,统计新生血管数目检测其生物相容性,检测外周血中调节性T淋巴细胞的比例。结果:实验组DNA含量较对照组下降超过95%(P<0.05);DAPI染色未见明显细胞核,扫描电镜及HE染色观察到实验组保留大量细胞外基质;SDS残留远低于生物安全水平;血管造影显示实验组血管分支完整、清晰,新生血管数明显多于对照组(P<0.05)。动物实验结果示血管再生数目多且调节性T淋巴细胞比例增高(P<0.05)。结论:提供了一个制备具有良好生物相容性且支架结构完整的去细胞皮瓣的方法,去细胞皮瓣具有低排斥和较好生物相容性的特点。

Objective:To prepare decellularized skin flaps by auxiliary shaking method and test their biocompatibility.Methods:After taking the skin flap of the lower abdominal wall of adult rats to remove blood,a magnetic stirrer was used to perform auxiliary shaking to remove cells.The biological characteristics of the harvested scaffolds were obtained by quantitative analysis of DNA,detection of SDS residues,and scanning electron microscopy.Then take the decellularized skin flap specimens embedded subcutaneously on the back of adult SD rats,count the number of new blood vessels to detect its biocompatibility,and the proportion of regulatory T lymph in the peripheral blood.Results:Compared with the control group,the DNA content of the decellularized group decreased by more than 95%;no obvious nucleus was seen in DAPI staining;scanning electron microscope and HE staining showed that a large amount of extracellular matrix remained in the decellularized group;SDS residues were far below the biological safety level;angiography showed that the branches of blood vessels were complete and clear,and the number of new blood vessels was significantly higher than that in the control group(P<0.05).The results of animal experiments showed that in decellularized group the number of blood vessels was regenerated and the proportion of regulatory T lymphocytes increased(P<0.05).Conclusion:A protocol for preparing decellularized skin flaps with good biocompatibility and complete stent structure is provided.