一种新型大鼠胰腺去细胞化支架的制备研究

摘要

目的 通过经胃左动脉途径灌注,选择胰腺体尾部作为灌注主体,应用各种去细胞化灌注技术,以制备新型天然胰腺去细胞化生物支架,并全面评价其理化特性,以期为胰腺组织再生工程的研究提供良好的应用载体.方法 选择健康成年雄性SD大鼠,离体获取胰体尾部作为灌注主体,采用胃左动脉作为灌注入路,通过物理冻融、酶解法及曲亚通(TritonX-100)为主辅以十二烷基硫酸钠(SDS)的洗脱剂组合进行顺行灌注,获取胰腺去细胞支架.并通过大体形态观察、美兰染色、组织病理学观察、基因组DNA定量分析、电镜观察、胶原蛋白成分鉴定、免疫原性分析等对去细胞化后的胰腺支架进行全面的评价.结果 本研究方法可成功制备肉眼透明的胰腺去细胞化支架,组织学检测和电镜观察均表明去细胞支架内无细胞残留,内部脉管网络和空间结构保存完整;免疫荧光分析表明去细胞化后支架的细胞外基质成分保留得较为完整,且支架DNA残留量为(42.3±10.6)ng/mg,不足正常胰腺的5%;皮下移植实验证实获得的去细胞化支架具有良好的组织相容性.结论 以胰腺体尾部作为灌注主体,经胃左动脉途径灌注的策略能够成功制备一种新型的去细胞化胰腺支架,并且支架的基质成分和理化特性较完整地保留,能够为胰腺组织工程研究提供一种良好的载体选择.%Objective To prepare a new type of natural pancreatic decellularized scaffold through decellu-larized infusion from left gastric artery and to evaluate its physicochemical properties, in order to provide an alternative platform for pancreatic tissue engineering. Methods Adult male Sprague-Dawley rats were use in this experiment. Detergents TritonX-100 supplemented with sodium dodecyl sulfate were perfused into the pancreas through the left gastric artery after physical freeze-thaw and enzymatic hydrolysis. After decellularization, scaffold were evaluated by general observation, methylene blue staining, HE staining, quantitative analysis of genomic DNA, transmission and scanning electron microscopy (TEM and SEM), collagen identification and immunogenicity analysis. Results This study successfully prepared a transparent pancreatic scaffold. Histological examination and electron microscope showed that there was no cell residual in it, and the internal vascular network and spatial structure were well preserved. Immunofluorescence analysis showed that the extracellular matrix components of the scaffolds were completely preserved. DNA content was confirmed to be (42.3±10.6) ng/mg, less than 5% compared to the normal tissue. Subcutaneous transplant experiments showed that the decellularized scaffolds had good histocompatibility. Conclusion The new decellularized pancreatic scaffold could be successfully prepared with its physical and chemical properties preserved under the perfusion strategy, and it may be used as an alternative platform for pancreatic tissue engineering study.