文章摘要
朱沙俊,徐天鑫,王 雷,钱海鑫,王志伟.小鼠胰岛与肝脏脱细胞支架三维共培养的实验研究[J].南京医科大学学报,2015,(12):1685~1690
小鼠胰岛与肝脏脱细胞支架三维共培养的实验研究
Three-dimensional culture of mouse pancreatic islets and liver-derived perfusion-decellularized bioscaffold for potential clinical practice
投稿时间:2015-05-03  
DOI:10.7655/NYDXBNS20151203
中文关键词: 糖尿病  脱细胞  生物支架  胰岛  移植
英文关键词: diabetes  decellularization  bioscaffold  islets  transplantation
基金项目:国家自然科学基金资助(81471801)
作者单位
朱沙俊 南通大学附属医院普外科,江苏 南通 226001
苏州大学附属第一医院普外科,江苏 苏州 215001 
徐天鑫 南通大学附属医院普外科,江苏 南通 226001 
王 雷 南通大学附属医院普外科,江苏 南通 226001 
钱海鑫 苏州大学附属第一医院普外科,江苏 苏州 215001 
王志伟 南通大学附属医院普外科,江苏 南通 226001 
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中文摘要:
      目的:观测原代小鼠胰岛在小鼠肝脏脱细胞支架上的生长及功能,探讨组织工程学治疗糖尿病的新方法。方法:运用灌注法制备结构完整的全肝脏脱细胞支架并检测,运用胶原酶P灌注消化法获取小鼠胰岛细胞并行结构和功能完整性分析;将分离纯化的原代小鼠胰岛经门静脉灌入全肝脏脱细胞支架,在三维培养体系培养后进行相关检测。结果:肝脏脱细胞支架检测未见细胞结构,胶原结构保存完整。支架内剩余DNA定量检测结果为(38 ± 11)ng/mg dsDNA,生物相容性良好。双硫腙(dithizone,DTZ)染色胰岛呈现特异性猩红色,葡萄糖刺激实验提示在高糖组中培养的原代小鼠胰岛胰岛素分泌功能明显高于低糖组,差异具有统计学意义(P < 0.01);将胰岛移植入肝脏脱细胞支架培养5 d后胰岛定植,且胰岛素基因表达显示脱细胞支架三维培养中胰岛素表达水平与平面培养相比,差异具有统计学意义(P < 0.01)。结论:肝脏脱细胞支架三维培养体系中的胰岛细胞比传统平面培养具有更佳的细胞活力及更有效的胰岛素分泌功能。
英文摘要:
      Objective:To observe the growth and function of primary mouse islets in the liver decellularized bioscaffold(LDB),and explore the new method of tissue engineering for treatment of diabetes. Methods:We perfused the whole mouse liver using detergent through hepatic portal vein in a continuous way and manufactured the whole LDB with the complete structure. The collagenase P was perfused into the mouse common bile duct,and the number of the isolated isles was counted and the integrity of the islets’ structure and function were analyzed. The isolated primary mouse islets were transplanted into the LDB and cultured for 5 days in the three-dimensional culture system. Then we appled HE staining,insulin immunohistochemical analysis and fluorescence quantitative polymerase chain reaction(PCR). Results:The LDB maintained their original round shape after decellularization and demonstrated a complete lack of nuclear staining and blood vessel. Collagenase 1 fluorescent staining showed that the collagen structure intact. The DNA quantification showed(38±11)ng/mg dsDNA in dry weight of ECM. In vivo biocompatibility test of the LDB showed that there didn’t exist any pathological presentation. The result of dithizone(DTZ)staining showed that the staining islets had the scarlet color Glucose stimulation test showed that high quality islets results in a greater insulin stimulation at high glucose concentration than that observed at low glucose concentration (P < 0.01). Insulin immunohistochemistry examination showed that the LDB parenchyma in transplanted area had brown stain. Insulin gene expression displayed the expression level of insulin in LDB was two times greater than the level of plate culture with statistical significance(P < 0.01). Conclusion:In LDB 3D culture system,islets showed better cell viability and more effective insulin secretion function than traditional plate culture.
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