两种不同去污剂制备肺去细胞支架的对比

摘要

Objective:To compare the effects of ionic detergent SDS with sodium deoxycholate so as to find a suitable method to obtain lung scaffolds which simulate natural differentiated extracellular matrix that promote cellular adhesion and differentiation.Methods:Sixty SD rats were randomly divided into normal group,SDS group and sodium deoxycholate group with 20 rats in each group.The heart-lung association was taken in each group.In both acellular groups,the rats were acellulared via the right ventricle and the pulmonary artery catheter was placed.In group A,perfusion was made with heparin,Triton X-100 and SDS solution followed by deionized water.Perfusion in group B was made with heparin,Triton X100,sodium deoxycholate solution and deionized water.Each scaffold was observed with scanning electron microscope.H-E staining and Masson trichrome staining were used to detect the residual cells and the nuclear component.Immunofluorescence staining was used to detect the residual quantity of laminin and fibronectin,and the DNA content was measured.Results:Scanning electron microscopy showed that the alveolar structure of SDS group scaffolds were more complete than sodium deoxycholate group.H-E staining showed no obvious cell and nuclear component remained after decellularization of SDS group.Residual nuclear component was found in sodium deoxycholate group.Masson staining showed that SDS group scaffolds had more elastic fibers than sodium deoxycholate group scaffolds.Immunofluorescence showed SDS group scaffolds kept more laminin and fibronectin protein than sodium deoxycholate group.The content of DNA of SDS group (35.28±8.20 ng/mg) was much lower than that in sodium deoxycholate group (52.33 ± 7.67 ng/mg) and the difference was statistically significant.Conclusion:Both the two kinds of reagents can eliminate the nuclear component in the lungs while maintaining extracellular matrix,but SDS perfusion is proved to be better than sodium deoxycholate perfusion.%目的:比较离子型洗涤剂十二烷基磺酸钠(SDS)与脱氧胆酸钠针对肺去细胞支架制备的性能,寻找一种合适的肺去细胞方法以得到模拟可以促进细胞附着和分化天然细胞外基质的肺功能架构.方法:SD大鼠随机分成正常对照组、去细胞SDS组、去细胞脱氧胆酸钠组,分别取心、肺联合体,两组去细胞组均经右心室置入留置针至肺主动脉.去细胞SDS组使用肝素、Triton X-100、SDS溶液和去离子水依次灌注;去细胞脱氧胆酸钠组使用肝素、Triton X-100、脱氧胆酸钠溶液和去离子水依次灌注.各组分别采用扫描电镜观察支架结构;H-E染色、Masson三色染色观察残留细胞、细胞核成分;免疫荧光染色观察肺组织层黏连蛋白、纤维连接蛋白保留情况,并进行DNA含量测定.结果:扫描电镜显示去细胞SDS组支架肺泡结构较去细胞脱氧胆酸钠组完整;H-E染色显示去细胞SDS组肺支架未见明显细胞及细胞核成分残留,去细胞脱氧胆酸钠组可见少量细胞核成分残留;Masson染色显示去细胞SDS组弹性纤维保留优于去细胞脱氧胆酸钠组;免疫荧光显示去细胞SDS组层黏连蛋白和纤维连接蛋白结构较去细胞脱氧胆酸钠组完整,保留度较高;去细胞SDS组DNA含量为(35.28±8.20) ng/mg,明显低于去细胞脱氧胆酸钠组(52.33±7.67) ng/mg,差异有统计学意义.结论:两种去污剂均可有效清除肺内细胞成分,较好地保留细胞外基质,其中SDS溶液灌注法优于脱氧胆酸钠溶液灌注法.