摘要目的 制备大鼠肝脏脱细胞支架及原代培养大鼠骨髓源性内皮祖细胞,用内皮祖细胞血管化大鼠肝脏脱细胞支架.方法 采用灌注法制备大鼠肝脏脱细胞支架,苏木素-伊红(HE)染色法、免疫组织化学检测脱细胞支架结构及成分;原代培养并鉴定大鼠骨髓源性内皮祖细胞,并通过CD31、CD133、血管内皮生长因子(VEGF)免疫荧光鉴定;构建循环灌注培养体系;采用经门静脉途径将内皮祖细胞种植于脱细胞支架内,行体外循环灌注培养,通过HE和CD31检测内皮祖细胞在支架内生长情况.结果 采用灌注法获得大鼠肝脏脱细胞支架,HE未见细胞核成分,同时可见细胞外基质成分保留,免疫组织化学显示I型胶原蛋白、层粘连蛋白保留;原代培养的大鼠骨髓源性内皮祖细胞CD31、CD133、VEGF免疫荧光阳性;构建出由蠕动泵、氧合器、培养瓶、输送管道组成的循环灌注培养体系;成功将内皮祖细胞通过门静脉途径种植到脱细胞支架内,HE、CD31显示内皮祖细胞定植于脱细胞支架管腔内壁.结论 通过循环灌注培养能够成功将原代大鼠内皮祖细胞种植于大鼠肝脏脱细胞管腔内壁.

abstractsObjective To engineer intact whole rat liver decellularized scaffolds and repopulated bone marrow-derived endothelial progenitor cells (EPCs) into the scaffolds by continous perfusion technology thus providing experimental support for the vasculation of decellularized liver scaffolds in liver engineering.Methods Decellularized liver scaffolds were obtained by perfusing method.The composition and structure was examed by hematoxylin-eosin staining (HE) and immunohistochemistry.Bone marrow derived EPCs were characterised by immunofluorescence of CD31,CD133 and Vascular endothelial growth factor (VEGF).EPCs were recellularized into the decellularized scaffolds by portal-vein infusion method and cultured by the dynamic circulation perfusion device.After cultivation,HE staining and immunofluorescence of CD31 were conducted to observe the growth situation of EPCs in the scaffolds.Results The rat decellulariezd liver scaffolds were successfully obtained by perfusion method.Histological staining demonstrated the remove of cellular component and the reservation of extracellular cell matrix.Immunohistochemistry staining demonstrated the retention of collagen I and laminin.The primary cultured EPCs were postive for CD31,CD133 and VEGF.The circulation perfusion device was composed of a peristaltic pump,oxygenator,chamber and the convey tubes.HE staining and immunofluorence revealed that EPCs can be located around the blood vessel wall.Conclusion The primary cultured EPCs can grew around the blood vessel wall of the decellularized liver scaffolds by circulation perfusion method.