去细胞组织工程异种瓣膜支架制备方法的比较研究

Studies on different decellularization procedures for heart valve tissue engineering

目的:探讨异种组织工程心脏瓣膜生物支架的最佳制备方法。方法:将16个新鲜猪主动脉瓣分为4组,每组4个,对照组未脱细胞,实验组分别为NaCl-SDS(十二烷基硫酸钠,sodium dodecyl sulfate,SDS)法脱细胞组、胰蛋白酶法脱细胞组、曲拉通法脱细胞组,观察其形态学和免疫组织化学变化。结果:NaCl-SDS法脱细胞处理的瓣膜内皮细胞去除不彻底,细胞外基质的三维网状结构虽完整但纤维模糊,肿胀。胰蛋白酶法处理的瓣膜内皮细胞基本去除,但瓣膜支架结构改变很明显,胶原纤维肿胀,边缘毛糙,纤维网状间隙增宽不规则。曲拉通法处理的瓣膜内皮细胞去除彻底,支架结构保存完好。NaCl-SDS法、胰蛋白酶法和曲拉通法脱细胞后的瓣膜均存在一定的免疫原性,但用胰蛋白酶法和曲拉通法脱细胞后的瓣膜支架的免疫原性较NaCl-SDS法显著降低。结论:应用曲拉通法制作的瓣膜支架去内皮细胞较为彻底,不改变瓣叶支架结构,而且免疫原性小,因此曲拉通法是一种良好的组织工程瓣膜支架制作方法。

Objective：The purpose of this study is to seek better method of making tissue engineering of heart valves by testing different decellularization procedures for their potential of cell removal and their ability to preserve the matrix. Methods：Specimens of porcine aortic valves were randomly divided into the control groups and the decellularized groups. The porcine aortic valves of decellularized groups were treated with either NaCL and sodium-dodecyl-sulfate（SDS） or trypsin or Triton-X100 with specific concentration. Tissue samples were then processed for Hematoxylin Eosin and scanning electron microscopy and immunohistochemistric staining for major histocompatibility complex class Ⅰ（MHC-Ⅰ） antigen. Results：NaCL and SDS achieved only incomplete decellularization, the main components of extracellular matrix were reserved completely but the fibrous components become swelling. In contrast, trypsin removed cells completely but caused strong structural alterations. Treatment with Triton X100 achieved both complete decelluarization and preservation of the matrix structure. The decellularized valves were reserved varied level of immunogenicity. MHC-Ⅰ was decreased remarkably in trypsin and Triton-X100 groups compared with NaCL and SDS group. Conclusion： The decellularization method by Triton X100 is effective. The decellularized porcine aortic valves reserve matrix structure and have lower immunity which can be used as an ideal valve for developing tissue engineering valve.