与胎鼠胰腺干细胞共培养保护大鼠胰岛活性  

Coculture with fetal rat pancreatic stem cells to protect the viability of islet

作　　者：张育森[1] 周汉新[1] 张悦[1] 余小舫[1] 李富荣[2] 

机构地区：[1]暨南大学第二临床医学院普外科,广东省深圳市518020 [2]暨南大学第二临床医学院临床研究中心,广东省深圳市518020

出　　处：《中国组织工程研究与临床康复》2008年第12期2313-2316,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广东省自然科学基金资助项目(6027540);深圳市科技计划项目(200601015)~~

摘　　要：目的：如何保护胰岛功能一直是胰岛移植中一个重要的研究内容。很多研究表明,干细胞除了自身具有较强的增殖分化能力之外,还可通过分泌一些细胞因子或者通过细胞-细胞接触、融合等方式促进损伤组织的修复,维持正常组织细胞功能。本实验利用胎鼠胰腺干细胞与大鼠胰岛共培养,观察其在保护胰岛功能、延长胰岛体外存活时间方面的作用。方法：实验于2006-07/2007-04在深圳人民医院中心实验室完成。①实验材料：选取体质量500g、孕16d的雌性SD大鼠1只,10周龄、体质量200～250gSD大鼠20只,雌雄不限,均由广东省实验动物中心提供,实验过程中对动物处置符合动物伦理学标准。②实验方法：分离纯化孕16dSD大鼠胎鼠的胰腺干细胞,并应用免疫细胞化学法及流式细胞术鉴定;采用胶原酶Ⅴ消化、不连续密度梯度法分离纯化SD大鼠胰岛,并应用双硫腙鉴定。按随机对照表法将胰岛培养分单纯胰岛培养、胰岛与胰腺干细胞联合培养两组,15孔/组。③实验评估：观察胰岛形态变化及胰岛存活率,应用酶联免疫吸附法检测胰岛素分泌量并计算刺激指数。结果：①胎鼠胰腺干细胞培养传代3代后免疫细胞化学可见巢蛋白阳性细胞,流式细胞术测定巢蛋白阳性细胞含量占74.1%。②单纯培养组培养3d时,胰岛生长状况较好,轮廓清楚,培养7d时,胰岛结构变松散,边界不清,培养超过14d后,胰岛结构均已破坏。联合培养组培养3d时,胰岛生长良好,大部分胰岛悬浮生长,部分胰岛与贴壁干细胞松散黏附,培养7d后大部分胰岛仍完整,培养14d时仍可见完整胰岛。③联合培养组培养7,14d时胰岛存活率明显高于单纯培养组（P〈0.01）;④高糖刺激下联合培养组培养7d时胰岛素分泌刺激指数明显高于单纯培养组（P〈0.01）。结论：胎鼠胰腺干细胞与胰岛联合培养可以明显延长胰岛体外存活时间,并�AIM： How to maintain the function of islets is one of important aspects in islet transplantation. Recent researches indicate that the stem cells can promote the repair of injured tissue or cells and maintain the normal function of tissue through cytokines, cell-cell contact or cell confluence. Our experiment is aimed to observe the effect of fetal rat＇s pancreatic stem cells （PSCs） on prolonging the survival time of islet and maintaining the function of islet in vitro by coculture of rat＇s islet with PSCs. METHODS： Experiments were performed at the Central Laboratory of Shenzhen People＇s Hospital from July 2006 to April 2007. （1） One female SD rat （500 g） on its 16 gestational days and 20 adult SD rats aged 10 weeks （200-250 g）, irrespective of gender, were provided by Guangdong Experimental Animal Center. Animal intervention met the animal ethical standards, （2）The PSCs were harvested from pancreatic rudiments of rat embryos on 16 gestational days and identified by immuocytochemistry and flow cytometry. The pancreas of SD rats were digested with collagenase V, which was injected into the common bile duct, and then the islets were separated with discontinuous Ficoll gradients.The islets were randomly divided into two group： one for islet culture alone, another for islet and PSCs culture together, with 15 holes in each group.（3）The status and survival rate of islets in both groups were observed. The amount of insulin secretion was measured by enzyme-labeled immunosorbent assay （ELISA） and insulin stimulation index was calculated in the insulin release test. RESULTS： （1）The immuocytochemistry showed that the PSCs expressing nestin; and the flow cytometry showed that 74.1% of cells expressed nestin after 3 generations.（2）In group islets culture alone the islets grew well and the body was intact in 3^rd day. Since the 7^th day, the islets became loose and boundary was obscure. The structure of islet was almost destroyed in 14^th day. Correspondingly, most of islets we

关 键 词：胰腺干细胞 胎鼠 共培养 胰岛 活性 

分 类 号：R394.2[医药卫生—医学遗传学]