制备犬胸主动脉脱细胞血管支架的新方法

New method to prepare good acellular matrices of canine thoracic aortas

目的 研究制备犬胸主动脉脱细胞血管支架的新方法,制备出理想的犬胸主动脉脱细胞基质,从而为构建组织工程血管提供支架材料.方法 在无菌条件下从成年比格犬体内取出胸主动脉血管段（28根）,随机分成4组：A组血管段设为正常对照组;B组血管段置于-70℃的冰箱和4℃冰箱内反复冻融2次;C组血管段在0.1％的SDS溶液中持续震荡1d;D组血管段经历反复冻融后置于1％ Triton X-100的PBS溶液和1μmol/L PMSF的混合液中常温下震荡2d,于0.01％的SDS溶液中持续震荡1d,最后加入核酸酶Dnase 0.2 mg/L和Rnase0.02 mg/L消化24h.四组血管段经历不同的过程后,全部从大体形态、光镜、电子显微镜观察、力学性能测试、免疫组化等角度进行检测并做统计学分析.结果 0.1％ SDS法虽能完全脱除细胞,但制备的脱细胞基质弹力纤维排列紊乱,部分发生断裂,且不能保持良好的形状和张力强度,管腔有不同程度的塌陷;反复冻融＋1％ Triton X-100＋PMSF＋0.01％ SDS法不仅能完全脱除细胞,保持基质纤维的正常排列结构,而且制备的脱细胞基质能保持良好的形状和力学性能,管腔无塌陷.结论 联合应用反复冻融、Triton X-100、PMSF和SDS的方法能够将犬胸主动脉的细胞成分除去,是一种制备脱细胞血管支架的有效方法.

Objective This paper is to explore a new method to remove all the cells from canine thoracic aortas for preparing good acellular matrices of canine thoracic aortas and then providing scaffolds for constructing tissue-engineering blood vessels.Methods Twenty eight canine thoracic aortas were divided into four groups randomized：group A（n=7） was normal canine thoracic aortas,group B（n=7） was frozen and thawed repeatedly for two times,group C （n=7） was treated with 0.1％ SDS,group D （n=7） was frozen and thawed repeatedly for two times and then treated with phosphate-buffered saline（PBS） containing 1％Triton X-100 and 1 μ mol/L phenyl methyl sulfonyl fluoride （PMSF） for two days,finally treated with 0.01％ SDS for one day.Treated specimens were observed with naked eyes,optical microscope and scanning electron microscope,their mechanic properties were measured and compared among them.Results Although method of 0.1％ SDS could entirely remove the cells of samples,the elastic fibers of vessel wall were in a great mess or destroyed,the acellular matrices prepared with it did not maintain shape and strain.The method in group D not only entirely removed the cells of samples and maintained the normal structure of matrix fiber in samples,but also maintained good shape and mechanical properties of the acellular matrices prepared.Conclusion The method in group D is a better and new method for preparing acellular matrices of canine thoracic aortas.