两种肝脏全器官脱细胞基质制备方法的比较

Comparison of two preparation methods of liver whole-organ decellularized matrix

目的对两种肝脏全器官脱细胞基质制备方法进行比较,为筛选更为有效的制备方法提供依据。方法选取两种脱细胞方案[分别以十二烷基硫酸钠(SDS)和Triton X-100+SDS(Tx+SDS)为主要洗涤剂]制备肝脏全器官脱细胞基质,制备完成后进行三维形态、脉管结构及组织学、免疫荧光染色观察,并对脱细胞基质中的DNA残留量、胶原含量及生长因子[肝细胞生长因子(HGF)、碱性成纤维细胞生长因子(bFGF)]含量进行检测。结果两种方法均成功制备出肝脏全器官脱细胞基质。组织学和DNA含量检测显示两种方案均可成功移除细胞和细胞核组分,保留细胞外基质(ECM)蛋白;Tx+SDS方案可以维持肝脏的天然脉管结构,而SDS方案则会损坏脉管结构;与SDS方案比较,Tx+SDS方案能保留更多的ECM蛋白成分(7.76±0.15μg/mg vs 4.23±0.05μg/mg),更多的HGF含量(44.5±2.9ng/g vs 11.3±1.8ng/g)和bFGF含量(15.9±0.4ng/g vs 6.1±0.7ng/g)。结论以Tx+SDS为主要洗涤剂的脱细胞方法成功制备出了较好的肝脏全器官脱细胞基质,可为肝组织工程和再生医学提供优良的支架材料。

Objective To compare the two different decellularization methods for preparation of liver whole-organ decellularized matrix for providing an experimental basis for screening more effective method. Methods Liver whole-organ decellularized matrix was prepared by two different decellularization methods using sodium dodecylsulfate （SDS） and Triton X-100＋SDS （Tx＋SDS） as main detergent respectively. The three-dimensional shape, vascular structure, histological results and immunofluorescence images were observed, and the contents of residual DNA, collagen and growth factors （HGF, bFGF） were determined by ELISA. Results Liver whole-organ decellularized matrix was successfully prepared by both methods, as the cellular and nuclear components were removed successfully and the extracellular matrix （ECM） protein was preserved. The Tx＋SDS method preserved the vascular structure of the liver, and the SDS method resulted in damage to the vascular structure. Compared with SDS method, the Tx＋SDS method preserved more ECM protein, including almost 100% of collagen, and 58.5% of HGF and 42.5% of bFGF. Conclusion The liver whole-organ decellularized matrix could be well prepared by Tx＋SDS method, and it may provide an effective scaffold for the study of tissue engineered liver and regenerative medicine.