聚乙醇酸生物支架与胰岛的共培养  

Culture of islets on a polyglycolic acid scaffold

作　　者：宋纯[1,2] 黄玉东[1] 侯艳[3] 宋一民[4] 谢万均[5] 黄任平[2] 魏争[2] 张增岭[2] 石于波[2] 宋春芳[2] 

机构地区：[1]哈尔滨工业大学应用化学系,黑龙江省哈尔滨市150001 [2]哈尔滨医科大学附属第一医院普通外科,卫生部细胞移植重点实验室,黑龙江省哈尔滨市150001 [3]哈尔滨医科大学统计学教研室,黑龙江省哈尔滨市150086 [4]北京协和医科大学附属协和医院普通外科,黑龙江省哈尔滨市100730 [5]哈尔滨医科大学附属第一医院内分泌科,黑龙江省哈尔滨市150001

出　　处：《中国组织工程研究与临床康复》2009年第16期3119-3123,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30570931)~~

摘　　要：背景:以往的研究表明,胰岛细胞体外培养的时间不宜过长,否则会出现纤维组织过度增生,使胰岛大量死亡,而导致移植失败,所以改善胰岛细胞体外培养环境是非常重要的。目的:将大鼠胰岛种植在聚乙醇酸生物支架上,并进行胰岛与支架共培养,观察支架上胰岛细胞的生长状态、胰岛细胞的形态和胰岛细胞的分泌功能,以寻找胰岛细胞体外生存的良好环境。设计、时间及地点:体外培养,对比观察实验,于2005-06/2008-12在哈尔滨医科大学附属第一医院,卫生部细胞移植重点实验室完成。材料:聚乙醇酸支架纤维直径15μm,网孔100～150μm,空隙率为97%,厚度为1.0～2.0mm。细胞培养前将支架浸泡入体积分数为75%乙醇溶液中30min,PBS冲洗3次,吹干后放入2g/L多聚赖氨酸中浸泡30min,消毒后备用。方法:采用胶原酶Ⅴ分离和消化大鼠胰岛,对照组胰岛加入含体积分数20%胎牛血清、10g/L青霉素-链霉素-两性霉素、10mmol/L Hepes的RPMI-1640培养液;支架组胰岛放在多聚赖氨酸包被的聚乙醇酸支架上,再加入同样的培养基,每个培养皿中细胞浓度为0.6×103,将两组细胞置于体积分数5%CO2、37℃的恒温培养箱中培养。主要观察指标:应用双硫腙胰岛特异性染色,计算胰岛的数量及检测胰岛的纯度;MTT法检测胰岛的成活率;放射免疫法测定胰岛细胞培养上清液中的胰岛素含量;并用扫描电镜对支架上胰岛的黏附生长进行观察。结果:分离和消化后的胰岛被双硫腙染成红色,胰岛外分泌腺不着色,胰岛纯度≥85%;MTT及胰岛功能检测结果显示,支架组的胰岛成活率及胰岛功能明显增高,与对照组相比差异有显著性意义(P<0.05);扫描电镜观察,胰岛紧密黏附在聚乙醇酸支架上,支架上胰岛成三维立体生长。结论:聚乙醇酸支架与大鼠胰岛共培养可改善胰岛的成活率、胰岛的功能及形态,并使胰岛体外生存期延长。BACKGROUND： Previous studies have shown that islet cells cultured in vitro for a long period can induce hyperplasia of fibrous tissues, cause death of islet, and induce a failure of cell transplantation, so it is important to improve in vitro culture condition for islet cells. OBJECTIVE： By implanting islets on polyglycolic acid （PGA） scaffolds, to explore growth, morphology, and secretion of islet cells in order to an ideal in vitro survival environment. DESIGN, TIME AND SETTING： An in vitro contrast experiment was performed at the Key Laboratory of Cell Transplantation of the National Ministry of Public Health, the First Affiliated Hospital of Harbin Medical University between June 2005 and December 2008. MATERIALS： PGA scaffold （diameter 15 μm, mesh 100 150 μm, porosity 97%, and thickness 1.0-2.0 mm） was used in this study. The PGA scaffold was cultured in 75% alcohol solution for 30 minutes, rinsed with PBS three times, stored in 10 g/L poly-L-lysine for 30 minutes, and sterilized for preparation. METHODS： Islets were isolated and digested by Collagenase-V method. Islet in the control group was cultured with RPMI-1640 culture medium containing 20% fetal bovine serum, 2 g/L penicillin-streptomycin-amphotericin, and 10 mmol/L Hepes. While, islet in the scaffold group was implanted on poly-L-lysine-ceated PGA scaffold and cultured with the same RPMI-1640 culture medium at the density of 0.6×10^3. Cells in the two groups were incubated in 5% CO2 incubator at 37 ℃. MAIN OUTCOME MEASURES： Number and purity of islet were detected using dithizone staining; survival rate was measured using MTT method; islet content in supernatant was measured using radioimmunity method; adherent growth was observed under scanning electron microscopy. RESULTS： Islets isolated and digested were stained red by dithizone and no stain was observed in the islet exocrine gland. The purity of islets was ≥ 85%. The results of MTT and functional detection showed that the survival rate of islets in the scaffold group

关 键 词：聚乙醇酸 胰岛 双硫腙 扫描电镜 

分 类 号：R318[医药卫生—生物医学工程]