两种去污剂在制备脱细胞肺支架中的对比

Comparative assessment of two detergents for decellularized lung scaffolds

背景:制备脱细胞肺支架的一个困难在于必须充分去除细胞同时尽可能保留细胞外基质。灌注去污剂-核酸酶法已被广泛应用于脱细胞支架的制备。目的:对比脱氧胆酸钠和十二烷基硫酸钠两种去污剂用于制备脱细胞肺支架的效果。方法:取出24只雄性SD大鼠肺脏,分3组,正常对照组不进行任何干预;脱氧胆酸钠组灌注去污剂脱氧胆酸钠联合核酸酶,制备脱细胞肺支架;十二烷基硫酸钠组灌注去污剂十二烷基硫酸钠联合核酸酶,制备脱细胞肺支架;取3组肺组织,进行组织学染色、免疫荧光染色及DNA含量测定。将A549细胞分别接种于两组脱细胞肺支架上培养7 d,进行苏木精-伊红染色。将两组脱细胞肺支架分别埋于SD大鼠背部皮下,2周后进行移植组织Masson染色。结果与结论:(1)正常对照组肺组织布满细胞,两脱细胞肺支架均没有残留的细胞和细胞核,但脱氧胆酸钠组肺支架内肺泡形态结构较十二烷基硫酸钠组完整;两组肺支架的DNA含量均低于正常对照组(P<0.01);(2)培养7 d,两组肺支架上的细胞均是由边缘向组织中间生长、浸润,脱氧胆酸钠组肺支架内的细胞生长、浸润速度明显优于十二烷基硫酸钠组;(3)肺支架移植2周后,十二烷基硫酸钠组肺支架与周围组织融合欠佳,边界清楚,可见大量细胞浸润,分布较均匀,血管内血细胞清晰可见;脱氧胆酸钠组肺支架内新生血管数量明显多于十二烷基硫酸钠支架,并且新生血管直径更大;(4)结果表明与十二烷基硫酸钠相比,采用去污剂脱氧胆酸钠制备的脱细胞肺支架生物相容性更好,与周围组织融合速度更快,细胞浸润和新生血管形成更明显。

BACKGROUND:It is quite difficult to produce a decellularized lung scaffold,in which cells are removed and the extracellular matrix components(ECM)are preserved effectively.Perfusion of detergent-enzymes is an effective method with wide applications for decellularized lung scaffolds.OBJECTIVE:To investigate the effects of two detergents(sodium deoxycholate,SDC and sodium dodecyl sulfate,SDS)on the preparation of decellularized lung scaffolds.METHODS:Twenty-four male Sprague-Dawley rats were randomized into three groups:control group with no intervention,SDC group and SDS group.Decellularized lung scaffolds were prepared by perfusion of SDC or SDS combined with enzymes.The rat lung tissues in the three groups were taken for histological staining,immunofluorescent staining and DNA quantification.A549 cells were cultured and seeded onto the decellularized lung scaffolds for 7 days followed by hematoxylin-eosin staining.The decellularized lung scaffolds prepared by perfusion of SDC or SDS were subcutaneously implanted into the rat back,and the implants were retrieved and assessed by Masson staining after 2 weeks.RESULTS AND CONCLUSION:In the control group,there were abundant cells in the lung tissues.In the other two groups,the decellularized lung scaffolds were nearly transparent,and the morphology of the SDC scaffold was more close to the native lung.There were no residual cells and nuclei on the two scaffolds,and the DNA content in the SDS and SDC groups was significantly lower than that in the control group(P<0.01).At 7 days of culture,A549 cells cultured on the SDS and SDC scaffolds migrated from the edge to the center of the scaffold.Comparatively speaking,the migration ability of A549 cells on the SDC scaffolds was stronger,and there was obvious cell invasion and growth in the middle part of the lung.After 2 weeks of scaffold transplantation,the SDC implants poorly fused with the surrounding tissues,with a clear boundary,a large number of infiltrating cells distributed evenly,and intravascular blood cells were clearly visible;the number of new blood vessels with larger diameter in the SDC scaffold was significantly higher than that in the SDS scaffold.These findings indicate that the SDC scaffold has better biocompatibility than the SDS scaffold,which can fuse with the surrounding tissues faster and produce more infiltrating cells and new blood vessels.