不同脱细胞方法对无细胞真皮基质抗原成分的影响

Effects of different methods for decellularization on antigen component of acellular dermal matrix

目的:异体/异种真皮无细胞化可直接关系到移植后局部炎症反应-免疫反应程度和复合皮的质量,因而探索不同的脱细胞方法对无细胞真皮基质抗原成分的影响。方法:实验于1999-07/2000-07在第四军医大学动物实验外科完成。将人和小猪正常中厚皮肤经体积分数为0.002的戊二醛交联后,用1.25g/L胰蛋白酶、1.25g/Ldispase和高渗盐(3mol/LNaCl)分别与体积分数为0.005的TritonX-100依次或同时进行脱细胞处理24h,制成的脱细胞真皮基质(ADM),再通过组织学和免疫组化方法进行抗原性比较。结果:单独使用胰蛋白酶、dispase和NaCl处理能使表皮有效地移除,但真皮中仍有较多细胞残留,如将其与TritonX-100联合或再经Tri-tonX-100处理24h后,真皮组织内细胞相关抗原(HLA-ABC,HLA-DR,波形蛋白,结蛋白,肌动蛋白,Ⅷ因子相关抗原和细胞角蛋白)全部消失,并且细胞外基质成分(透明质酸、纤连蛋白、层粘连蛋白和Ⅳ型胶原)以及Ⅰ,Ⅲ和Ⅶ型胶原等抗原明显减弱。相比之下,戊二醛交联组和NaCl+TritonX-100处理的ADM浅层保留较多的Ⅳ型胶原、纤连蛋白和层粘连蛋白成分。各种方法制备ADM的抗原强弱依次为:NaCl(+TritonX-100)>dispase(+TritonX-100)>胰蛋白酶(+TritonX-100)。结论:采用胰蛋白酶联合去污剂制备ADM是一简单。

AIM:The decellularization of allogeneic or xenogeneic dermis may be directly related to the level of local inflammatory immunologic reaction and the quality of composite skin after grafting.The aim of this study was to compare the influence of different methods for decellularization on the antigen components of acellular dermal matrix(ADM).METHODS:The experiment was conducted in the Department for Experimental Animals,Fourth Military Medical University of Chinese PLA from July 1999 to July 2000.Human and porcine split thickness grafts were cross linked with 20 mL/L glutaraldehyde,then treated with 1.25 g/L trypsin,1.25 g/L dispase or 3 mol/L NaCl solution,which combined with or without 5 mL/L Triton X 100 respectively for 24 hours,respectively.Histochemical and immunohistochemical staining was performed to observe and compare the antigenicity of these ADMs.RESULTS:The epidermis was effectively removed alone with trypsin,dispase or NaCl solution,but there were still many cells left in the dermis.After the dermis was treated with trypsin,dispase or NaCl solution together or further with Triton X 100 for 24 hours,the cell relative antigens,such as HLA ABC,HLA DR,vimentin,desmin,actin,factor Ⅷ related antigen(FⅧ RA),and cytokeratin were removed sufficiently,and extracellular matrix components(hyaluronic acid,fibronectin,laminin and type Ⅳ collagen) and collagens of typeⅠ ,Ⅲ and Ⅶ were reduced markedly from the dermal matrix.However,type Ⅳ collagen,laminin and fibronectin were remained most in the superficial part of the ADM treated with glutaraldehyde or NaCl combined with Triton X 100.In comparison,the ADM treated with trypsin combined with Triton X 100 demonstrated the least antigenicity among these treatments.CONCLUSION:The ADM made by trypsin combining detergent for decellularization is simple,low cost,and effective to decrease the antigenicity of dermal matrix.