【摘要】 目的探讨脱细胞胆管细胞外基质(ECM)的制备方法及其替代胆总管缺损的可行性。方法采用4因素3水平9次实验的正交设计[L9(3~4)],切取活体犬的肝外胆管39段,每段长1 cm,其中27段按正交设计随机分为9组行脱细胞处理,试验重复3次。采用苏木素-伊红(HE)染色及计算机图像分析对 ECM 进行残余细胞成分计数,进行极差分析及方差分析,获得制备ECM 的最佳方案。另12段胆管按最佳方案制备成 ECM 并用于同种异体胆总管缺损修复实验。分别手术后2、4、12及24周取材观察缺损修复处组织再生情况。结果脱细胞后第7、9组未发现残余细胞,ECM 富含胶原,第9组胶原呈立体交叉交联。移植术后2周,ECM 内膜面见少量单层上皮细胞,有血管长入其中,ECM 中有少量成纤维和平滑肌细胞,吻合口处有较多炎性细胞浸润;4周ECM 管腔被上皮细胞覆盖,排列整齐,ECM 中有较多成纤维和少量平滑肌细胞,吻合口有少量炎性细胞浸润;12周吻合痕迹消失,ECM 中有较多成纤维、平滑肌细胞和少量纤维细胞;24周 ECM 中有以平滑肌细胞和纤维细胞为主,少量成纤维细胞,结构近似正常。结论制备 ECM 中3个关键条件的最佳水平为 A3B3C3,即在胆管脱细胞处理过程中采用0.4%胰蛋白酶、1.0%叠氮二苯基磷(DPPA)、40 U/ml DNA 酶。所得 ECM 应用于犬同种异体胆总管端端吻合修补,效果满意。更多还原

【Abstract】 Objective To study the preparation of the extracellular matrix(ECM)of bile duct and the feasibility of ECM substituting for bile duct defect in canines.Methods An orthogonal design [L9(3~4)] concerning three factors which contributes to the preparation of ECM was used in the experi- ment.1 cm long 39 segments of bile duct were obtained.Twenty-seven of them were decellularized accord- ing to the orthogonal design in 9 groups.The whole experiments were repeated for 3 times.All ECMs were haematoxylin-eosin stained and the number of the remain cellular elements compared by computer image a- nalysis to get the optimum process by which the 12 segments of ECMs were made from 12 segments of bile duct.Scanning and transmission electronic microscopy,α-actin staining and CK-18 staining were used to examine the decellularization.Subsequently,the ECMs were used as grafts to repair the defect of bile duct in 12 canines.The grafts were took out 2,4,12 and 24 weeks after operation.The regeneration was con- firmed by the haematoxylin-eosin staining and scanning electronic microscopy.Results Different decellu- larized processes led to ECMs differently in the number of remain cellular elements.There were no cellular element in the groups 7 and 9,but the fibers of collagen were more loosen in group 7 than in group 9.EC- Ms taken out from operated animals showed that the regenerating course of new tissue lasted since 2nd week to about 12th week postoperation.Condusion The best way to prepare the ECMs of bile duct is A3B3C3,namely 0.4% trypsin,1.0% DPPA and 40 U/ml DNase,and the ECMs obtained could be used in the repair to the defect of bile duct in canines.更多还原