去细胞处理对牛颈静脉带瓣管道细胞外基质骨架的影响

目的:探讨去细胞处理对牛颈静脉带瓣管道细胞外基质骨架成分及组织稳定性的影响,为确立构建组织工程化的人工血管技术提供依据.方法:采集新鲜牛颈静脉,筛选瓣膜功能良好的血管,采用曲那通X-100、胰蛋白酶/EDTA和DNase-I/RNase-A三步法去细胞处理,组织化学染色和透射电镜观察细胞外基质骨架成分(胶原和弹力蛋白)的变化.对新鲜和去细胞处理的牛颈静脉血管壁(n=10)分别进行弹力蛋白(Fastin弹性蛋白法)和羟脯氨酸(碱水解、分光光度法)含量测定;并测定处理前后的热皱缩温度和抗张强度以比较组织稳定性.结果:去细胞处理牛颈静脉血管壁光镜和透射电镜检测提示细胞及其成分完全去除,组织间隙增宽,但胶原成分较好保留,弹力蛋白部分减少及断裂.与新鲜组织比较,去细胞组羟脯氨酸含量相对增加[(25.73±2.97)mg/g vs.(29.25±2.99)mg/g,P＜0.05],弹力蛋白含量相对减少[(159.71±21.06)ng/g vs.(134.91±35.40)mg/g,P＜0.05],热皱缩温度降低[(72.50±0.53)℃ vs.(69.75±0.54)℃,P＜0.05],抗张强度也下降明显[(5.19±0.65)MPa vs.(3.13±0.94)MPa,P＜0.05].结论:去细胞处理对牛颈静脉细胞外基质骨架有一定损害,牛颈静脉的组织稳定性有所下降,得到的细胞外基质基本成分需经进一步交联处理才可以作为组织工程支架材料.

Effect of decellular treatment on the framework of extracellularmatrix in bovine jugular vein conduit

OBJECTIVE: To investigate the effect of decellular treatment on the framework constituents of extracellular matrix and tissue stability in bovine jugular vein conduit(BJVC),and to provide an evidence for tissue engineering of vascular prosthesis. METHODS: Bovine jugular veins were obtained fresh from a local slaughterhouse and were stored in chilled PBS. In the laboratory, any fat and loose connective tissue on the outer surface of the vessel was trimmed. BJVCs were decellularized by a 3-step extraction method as detergent Triton X-100(0.5%), Trypsin (0.025%)EDTA (0.02%), and DNase I(30kU/L) RNaseA(0.3g/L). Histological and transmission electron microscopy(TEM) techniques were used to study the framework constituents of extracellular matrix of treated the examples, and fresh tissues were used as controls. Tissue contents of hydroxyproline(alkaline hydrolysis method) and elastin(Fastin Elastin Assay) were assayed respectively in the fresh and decellularized groups (n=10). The vascular wall heat shrinking temperature and mechanical strength were measured to evaluate the tissue stability (n=10). RESULTS: Histochemical and TEM analysis of BJVCs treated with decellularization proved a complete removal of nuclear and other cell components. Tissue collagen was well kept,but elastin was partly lessened. Tissue content of hydroxyproline increased comparatively [(25.73+/-2.97)mg/g vs. (29.25+/-2.99)mg/g, P<0.05]and the elastin content obviously decreased[(159.71+/-21.06)mg/g vs. (134.91+/-35.40)mg/g, P<0.05]in the decellular treatment group compared with the control group. The heat shrinking temperature and tensile stress of decelluarized tissue were lower than those of the fresh tissue[(72.50+/-0.53)degrees C vs. (69.75+/-0.54)degrees C ,P<0.05],[(5.19+/-0.65)MPa vs. (3.13+/-0.94)MPa, P<0.05]. CONCLUSION: The basic framework of extracellular matrix in the decellularized BJVC is partly damaged and tissue stability is reduced. Decellularized BJVC should be further crosslinked before being used as a tissue engineering scaffold for clinical pulmonary artery graft.