背景：胆管损伤修复是腹部外科手术的难题，组织工程胆管是解决这一难题的理想方法，而构建性能优异的组织工程胆管支架是这一研究的关键。目的：观察猪骨髓间充质干细胞诱导分化的胆管内皮细胞与电纺纳米纤维的生物相容性。方法：将猪骨髓间充质干细胞于体外定向分化为肝干细胞，再进一步分化为胆管内皮细胞，并通过形态学及RT-PCR对分化完成的细胞加以鉴定。用静电纺丝方法制备聚乳酸-羟基乙酸纳米纤维膜，通过扫描电镜观察其形态，并测定其短期(2周)内体外降解率。将分化的胆管内皮细胞与纳米纤维复合培养，观察细胞在纳米纤维表面的黏附、增殖能力，荧光染色观察细胞形态及在材料表面的分布情况，扫描电镜观察细胞在纳米纤维表面生长形貌。结果与结论：骨髓间充质干细胞体外定向分化4周后，细胞呈现典型树枝状胆管内皮细胞形态学改变，并且有CK19的表达。扫描电镜照片显示电纺材料为连续的纳米纤维，纤维直径均分布在200-500 nm范围内，在2周内聚乳酸-羟基乙酸纳米纤维没有发生明显的降解。通过细胞黏附率计算、MTT 法检测、荧光染色及扫描电镜观察证明猪骨髓间充质干细胞诱导分化后的胆管内皮细胞与聚乳酸-羟基乙酸纳米纤维具有良好的生物相容性。%BACKGROUND:Repair of extrahepatic biliary tract injury is a difficult problem in the abdominal surgery. Tissue-engineered extrahepatic biliary tract is an ideal selection for this problem. Construction of tissue-engineered extrahepatic biliary tract with excelent performance is a key to related studies. OBJECTIVE:To investigate the biocompatibility of bile duct endothelial cels differentiated by porcine bone marrow mesenchymal stem cels with electrospun nanofibers. METHODS:Porcine bone marrow mesenchymal stem cels were induced toward biliary tract endothelial cels, which were then identified by morphology and RT-PCR. Polylactic-co-glycolic acid (PLGA) nanofiber membranes were prepared by electrospinning. The morphology was determined by scanning electron microscopy and the short-term (2-week)in vitro degradation rate was determined. Adhesion and proliferation of biliary tract endothelial cels on the nanofiber surface was analyzed by calculating the cel adhesion rate and MTT assay, respectively. Cel growth, morphology and distribution on the material surface were observed by fluorescence staining and scanning electron microscopy, respectively. RESULTS AND CONCLUSION: After 4 weeks of directed differentiation of bone marrow mesenchymal stem celsin vitro, cels showed typical morphology of dendritic bile duct endothelial cels and had the expression of CK19. Scanning electron micrographs showed that electrospun materials were continuous nanofibers with diameters between 200 and 500 nm. No significant degradation of the PLGA nanofibers was observed within 2 weeks. Based on the measured cel adhesion rate, MTT assay, fluorescence staining, and scanning electron microscopy, the differentiated cels possessed a good proliferative capacity on PLGA nanofibers. Bone marrow mesenchymal stem cels differentiated into bile duct endothelial cels in vitro. Materials prepared by the electrospinning method had a nanofiber structure, which did not significantly degrade within 2 weeks. Differentiated cels exhibit good biocompatibility with the nanofibers.
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