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目的探讨用猪小肠黏膜下层(SIS)作支架,复合大鼠胰岛体外构建组织工程胰腺的可行性。方法用改良的Ⅴ型胶原酶消化法方法分离纯化大鼠胰岛并培养,再与SIS进行体外复合培养。然后用相差显微镜、透射电镜和扫描电镜检查胰岛的形态,用丫啶橙/碘化丙啶(AO/PI)双染法观察胰岛的生长及细胞活性,用胰岛素释放试验评价胰岛细胞的分泌情况。采用独立t检验,分别比较在高糖、低糖条件下,SIS组与对照组胰岛素释放的差异有无统计学意义。结果胰岛在SIS材料上黏附、生长,细胞功能活跃,AO/PI双染法显示平均存活率92﹪,高糖下胰岛素释放量为(37.79±4.27)mmol/L,明显高于对照组[(28.69±3.58)mmol/L,t=12.678,P=0.000]。胰岛在SIS上成复层生长,复合培养时间明显延长。结论 SIS与胰岛在体外复合培养可以构建出类似生物膜的组织工程胰腺,为进一步研究体内胰岛移植奠定基础。
Objective To investigate the feasibility of constructing tissue-engineered pancreas in vitro using porcine small intestinal submucosa (SIS) as scaffold and compound rat islets. Methods The rat pancreatic islets were isolated and purified by a modified type Ⅴ collagenase digestion method and cultured in vitro with SIS. The morphology of islets was examined by phase contrast microscopy, transmission electron microscopy and scanning electron microscopy. The islet growth and cell viability were observed by acridine orange / propidium iodide (AO / PI) double staining. The insulin secretion was evaluated by insulin release assay . Independent t test was used to compare the difference of insulin release between SIS group and control group in high glucose and low glucose respectively. Results The pancreatic islets adhered and grew on SIS material with active cell function. The AO / PI double staining showed that the average survival rate was 92% and the insulin release rate was (37.79 ± 4.27) mmol / L under high glucose, which was significantly higher than that of the control group [ 28.69 ± 3.58) mmol / L, t = 12.678, P = 0.000]. Islets in the SIS stratified growth, significantly longer incubation time. Conclusion SIS and pancreatic islets can be constructed into tissue-engineered pancreas resembling biofilm in vitro and in vitro, so as to lay the foundation for further study of islet transplantation in vivo.