全肺去细胞生物支架的制备与鉴定

Preparation and identification of a whole lung decellularized scaffold

目的:通过聚乙二醇辛基苯基醚(Triton X-100)、十二烷基磺酸钠(SDS)灌注法制备大鼠全肺去细胞生物支架,并对其进行鉴定。方法:20只SD大鼠随机数字表法分成去细胞组与正常对照组,每组10只,分别取心、肺联合体,去细胞组经右心室置入留置针至肺主动脉,恒温37℃依次灌注肝素化PBS溶液、1%Tirton X-100、0.8%SDS及去离子水。进行DNA定量分析,HE染色及免疫荧光观察残留细胞及细胞核成分,丙烯腈-丁二烯-苯乙烯树脂(ABS)铸型观察肺内血管分布情况。结果:去细胞组DNA含量为(40.37±5.01)ng/mg,较正常对照组的(846.64±65.70)ng/mg下降95%以上;HE染色及免疫荧光染色见去细胞组肺生物支架保留了大量细胞外基质,未见明显细胞及细胞核成分残留;血管铸型标本显示去细胞组血管分布与正常对照组相仿,其分支完整、清晰。结论:Triton X-100、SDS灌注法可有效清除肺内细胞成分,较好地保留细胞外基质(ECM)和血管网络结构,是一种简便且较为理想的制备组织工程肺生物支架方法。

Objective： To prepare a whole lung decellularized scaffold that was perfused with TritonX-100 and SDS, and to perform preliminary identification. Methods： Twenty SD rats were divided into two groups of 10 case in each randomly （decellularization and control group）. Hearts and lungs were harvested from SD rats. In decellularization group, the arterial catheters were inserted through the aortopulmonary to establish channels for whole lung perfusion successively with heparinized PBS solution, 1% TirtonX-100, 0.8% SDS and deion- ized water in 37 ~C. The DNA concentration was determined after decellularization, and the scaffold and native lung were observed by HE staining, immunotluorescence and vascular cast. Results： Quantitative analysis of DNA content within the decellularized scaffold was （40.37-4-5.01） ng/mg, which showed a significant decrease compared to the native lung [（846.64：e65.70） ng/mg]. A lot of collagen fibers could be observed with HE and im- munohistochemistry stain but no visible cell nuclei remained after decellularization. Cast specimen showed that pulmonary arteries were still full and clear compared with the native lung. Conclusion： The method of perfusion with TritonX-100 and SDS can effectively remove all cellular components, and retain the extracellular matrix and vascular network structure well. It＇s a convenient and ideal preparation method on decellularized lung scaffold for tissue engineering.