猪主动脉脱细胞基质的简化制备及生物学评价  

SIMPLIFIED PREPARATION AND RELATIVE EVALUATION OF DECELLULARIZED PORCINE AORTIC SCAFFOLD

作　　者：张建[1] 吴英锋[1] 陈亮[2] 

机构地区：[1]首都医科大学宣武医院血管外科,北京100053 [2]中国药品生物制品检定所

出　　处：《中国修复重建外科杂志》2008年第3期364-369,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家高技术研究发展计划(863)资助项目(2006AA02A134);北京市科学技术委员会重大项目基金资助项目(H020920040330)~~

摘　　要：目的应用酶法制备猪主动脉脱细胞基质,并评价其生物学性能。方法取新鲜屠宰的16根肉猪降主动脉,长10～12cm,应用0.1%胰蛋白酶和0.01%EDTA于37℃震荡条件下脱除细胞成分,HE和Masson染色行脱细胞基质的组织学观察,扫描电镜和透射电镜观察超微结构改变。应用单轴拉伸方法比较脱细胞前和脱细胞后48、96和120h时材料两断端厚度、极限抗张强度(ultimatetensionstress,UTS)和断裂伸长率(strainoffailure,SOF),绘制应力-应变曲线。取成年杂种犬3只,体重20～30kg,雌雄不限。将脱细胞前后片状主动脉基质埋植于犬脊柱两侧皮下,分别于埋植后1、3和6周取材,行HE染色观察,参照半定量的Wakitani评分法,对取材时的大体形态、浸润细胞种类和数量、新生血管等指标进行比较,观察宿主炎性细胞反应和脱细胞基质变化。将犬内皮细胞种植于猪脱细胞基质,HE染色和扫描电镜观察细胞相容性。结果HE染色和电镜检查显示在96h可将新鲜猪主动脉细胞成分脱除,Masson染色显示细胞外纤维成分保留完整。脱细胞前后的动脉基质厚度、UTS和SOF差异均无统计学意义(P>0.05),但UTS显示降低趋势,SOF则呈现增加趋势,应力-应变曲线呈现力学强度降低和延展性增加的变化趋势。犬皮下埋植,各组脱细胞标本炎性细胞浸润明显减轻,6周时以成纤维细胞浸润为主,且基质中见新生毛细血管。半定量组织学评分,在大体观察、浸润细胞种类和数量方面与脱细胞前比较有统计学意义(P<0.05);新生血管数量比较,差异无统计学意义(P>0.05)。HE染色和扫描电镜显示种植的内皮细胞可在基质表面形成单细胞层。结论应用0.1%胰蛋白酶和0.01%EDTA持续振荡96h制备的猪主动脉脱细胞基质,在生物力学、免疫原性和细胞相容性方面可满足血管组织工程的需要。Objective To investigate the feasibility of applying enzymatic method to prepare decellularized porcine aorta and to evaluate its biomechanical properties, immunogenicity and cell compatibility. Methods 0.1% trypsin- 0.01% EDTA was applied to extract cells from porcine aorta under 37℃ continuously vibrating condition and its histology and microstructure were observed. The thickness, stress-strain curve, ultimate tension stress （UTS） and strain of failure （SOF） were compared before and after decellularization for 48, 96 and 120 hours under uniaxial tensile tests, respectively. The histological change was observed at 1, 3 and 6 weeks after the decellularized tissue was implanted subcutaneously in 3 dogs. According to the HE stains and a semi-quantitative Wakitani grading method, gross changes, category and amounts of infiltrated cells and neo-capillaries were compared between pre- and post-decellularization of porcine aortae. Endothelial cells from canine external jugular vein were seeded onto the decellularized patches to observe the cell compatibility. Results Microscopy and electron microscopies examination identified that cell components was completely removed from the fresh porcine aorta and Masson＇s trichrome showed that the structure of matrix （fibrins） was maintained intact at 96 hours using the decellularization method. There were no significant differences in the thickness, UTS and SOF between before and after decellularization （P 〉 0.05）. However, The UTS values showed a decrease tendency and SOF showed an increase tendency. The stress-strain curve also verified a decrease tendency in mechanical intensity and an increase one in ductility after decellularization. After implanting the acellularized matrix subcutaneously in canine, moderately lymphocyte infiltration was seen at the 1st week and the infiltration was replaced by fibroblasts accompanied by neocapillary formation at the 6th week. A semi-quantity histological evaluation showed that there were differences in gross observatio

关 键 词：组织工程 猪主动脉 脱细胞基质 生物力学 免疫原性 

分 类 号：R318[医药卫生—生物医学工程]