去细胞化技术在全肝生物支架建立中的应用

Decellularization technology application in whole liver reconstruct biological scaffold

目的通过去细胞化技术建立保留细胞外基质的完整肝脏支架，并对支架进行形态结构和保留成分鉴定。方法通过门静脉插管，依次灌注去垢剂曲拉通X-100（Triton X-100），十二烷基硫酸钠（SDS），并用磷酸盐缓冲液（PBS）洗脱残留去垢剂，对所得支架进行HE染色、门静脉及胆道铸型、扫描电镜、纤维成分染色鉴定等形态学观察。并将支架切成50μm的厚度后与C3A细胞系共培养，进行生物相容性验证。结果经本实验方案得到肝脏生物支架的成功率为75％。肝脏生物支架包膜完整，肉眼可见肝脏内Gllisson管道结构。HE染色示无细胞及胞核残存。管道铸型见胆道、门静脉完整，无铸型液溢出。扫描电镜示大量纤维网状结构存在。纤维成分染色见大量胶原纤维、弹力纤维存在。生物相容性实验初步显示该支架具备较好的生物相容性，可以作为生物支架材料应用。结论经本实验去细胞化过程，肝组织细胞可从细胞外基质中完全洗脱下来，并保留比较完整的肝脏管道系统。本研究获得全肝生物支架可以作为肝脏器官培养的基础支架。

Objective To explore an innovative method for preparing a whole-liver reconstruct scaffold with intact three-dimensional geometry, vasculature and bile duct by decellularization technology. Methods The portal vein was annulated and perfused sequentially with 1% Triton X-100 and 1% SDS for about 4 h, and then was perfused with phosphate buffered saline to dilute SDS residue. The retained structure was evaluated by histological analyses, including macroscopic, Hematoxylin-Eosin staining, Masson＇s trichrome staining, oreein staining and SEM. The liquid polymer preparation 8% - 10%, which was made of chlorinated poly vinyl chloride （CPVC as solute）, acetone （as solvent） and pigment, was injected into portal vein and bile duct to demonstrate the integrity of the portal vein and bile duct. The scaffold was cut into slices with the thickness of about 50 μm and co-cultured with C3A cell line. Results Macroscopic examination showed that the decellularized liver was transparent and intrabepatic Glisson＇s system could be observed. H＆E staining of slices of decellularized liver demonstrated no intact cells or nuclei existed. Masson trichrome staining revealed collagen retained. Orcein staining showed that there were elastic fibers. SEM showed the network of ECM was intact. C3A-to-scaffold co-culture revealed the scaffold of a good biocompatihility. Conclusion Peifusion with detergents through portal vein for liver decellularization is an efficient method to obtain a completely whole-liver scaffold for hepatic organ reconstruction.