肝脏脱细胞支架与MIN6细胞三维共培养移植治疗Ⅰ型糖尿病实验研究  

Three dimensional co-culture of MIN6 cells and liver-derived perfusiondecellularized bioscaffold for potential clinical practice

作　　者：朱沙俊[1] 王雷[1] 

机构地区：[1]南通大学附属医院普外科,江苏226001

出　　处：《交通医学》2015年第4期319-322,F0002,共5页Medical Journal of Communications

基　　金：国家自然科学基金资助项目(81471801)

摘　　要：目的:通过体外制备小鼠肝脏脱细胞支架并种植MIN6细胞,观测其定植生长并检测其功效发挥,为体外构建胰岛素分泌器官提供理论基础。方法:灌注法制备全肝脏脱细胞支架,将小鼠MIN6细胞种植入支架中于三维培养体系中培养,通过苏木精-伊红染色、胰岛素免疫组化分析及荧光定量聚合酶链反应检测其功效。检测MIN6细胞功能基因Insulin表达。将种植MIN6细胞的肝脏脱细胞支架移植入糖尿病小鼠体内,监测其功能发挥并初步评估其疗效。结果:体外成功制备肝脏脱细胞支架,在保留其原有脉管网络基础上,未见组织残留。HE染色显示肝脏脱细胞支架内没有细胞核染,且脉管结构保存完整。扫描电镜未见细胞结构,支架保存完整胶原纤维形态。MIN6细胞在脱细胞支架中三维培养后,检测胰岛素基因表达水平,结果和平面培养相比较,差异具有统计学意义(P<0.05)。将种植MIN6细胞的肝脏脱细胞支架移植入糖尿病小鼠体内能够较好的发挥降血糖作用。结论:通过将MIN6细胞种植入肝脏脱细胞支架并三维培养,可构建体外胰岛素分泌体系。Objective：To observe the growth and function of primary mouse islets in the liver decellularized bioscaf-fold （LDB）. At the same time, a new method of tissue engineering for treatment of diabetes was explored. Methods： The whole mouse liver was perfused by using detergent through hepatic portal vein in a continuous way and the whole liver de-cellularized bioscaffold was manufactured with the complete structure. The MIN6 cells cultured in the 3D bioscaffold. Then the hematoxylin-eosin （HE） staining, insulin immunohistochemical analysis and fluorescence quantitative polymerase chain reaction （PCR） were performed. qRT-PCR was conducted for the functional gene of INS1 and INS2 to further illuminate its function. The function of the recellularized pancreatic scaffold was monitored in vivo. Results：The liver decellularized bioscaffold, the extracellular cell matrix （ECM） of the LDB maintained their original round shape after decellularization and demonstrated a complete lack of nuclear staining as well as a complete network of blood vessel. Insulin gene expression was displayed, and the expression level of insulin in LDB was greater than that of the plate culture with statistical signifi-cance （P〈0.05）. MIN6 cells grown in the scaffold were better than those cultured on the culture dish. The recellularized pancreatic scaffold can control the blood glucose of the diabetic mice. Conclusion：LDB 3D culture system showed cell vi-ability with effective insulin secretion.

关 键 词：Ⅰ型糖尿病 肝脏脱细胞支架 移植 免疫组化染色法 实时定量聚合酶链反应 

分 类 号：R587.1[医药卫生—内分泌]