摘要
背景：损伤胰腺提取液含有胰腺再生过程的特异性生长因子，能够促进β细胞系增殖和胰岛素分泌，可能有助于胰岛再生和细胞分化。目的：观察损伤胰腺提取液对骨髓间充质干细胞分化为胰岛样细胞的影响。设计、时间及地点：细胞学体外观察，于2005-06/2007～03在解放军军事医学科学院生物工程研究所完成。材料：清洁级雄性Sprague-Dawley大鼠6只，由解放军军事医学科学院实验动物中心提供。活化素A、视黄酸、胰蛋白酶抑制剂为Sigma公司产品，链脲菌素为Sigma公司产品。方法：大鼠腹腔注射链脲菌素，2d后血糖浓度超过10mmol／L时取其胰腺组织，在含有胰蛋白抑制剂的磷酸盐缓冲液中制备匀浆，2次离心后取上清，即为损伤胰腺提取液。采用密度梯度离心法分离大鼠骨髓间充质干细胞，当传代细胞生长至70％～80％融合时，随机分为4组：对照组以体积分数为0．02的胎牛血清的低糖DMEM培养11d；活化素A＋视黄酸组以活化素A、视黄酸各自诱导24h，再以碱性成纤维生长因子、尼克酰胺、尼克酰胺＋exendin4各自诱导3d；活化素A＋视黄酸＋损伤胰腺提取液组在前组基础上添加损伤胰腺提取液；损伤胰腺提取液组单纯以损伤胰腺提取液诱导11d。主要观察指标：应用免疫细胞化学和反转录聚合酶链反应等方法对分化细胞表型进行检测，ELISA法检测细胞胰岛素的分泌水平。结果：活化素A＋视黄酸组、活化素A＋视黄酸＋损伤胰腺提取液组有较多的胰岛素阳性反应细胞出现，且后者形成的胰岛样结构较前者大而多;损伤胰腺提取液组可见较少的胰岛素阳性反应细胞及胰岛样结构。各诱导组均检测到胰岛素1mRNA的表达，培养上清中均可检测到胰岛素的分泌，且活化素A＋视黄酸＋损伤胰腺提取液组胰岛素分泌水平〉活化素A＋视黄酸组〉损伤胰腺提取液组（P〈0．05）。对照组各项指标均呈阴性表达。结论：基于前期活化素A、视黄酸及其他成熟因子的诱导方案基础上，损伤胰腺提取液对骨髓间充质干细胞分化为胰岛样细胞具有促进作用，能够提高诱导分化效率。
BACKGROUND： Extract of injured pancreatic gland contains specific growth factor, which can promote β cell proliferation and insulin secretion, resulting in insulin regeneration and cell differentiation. OBJECTIVE： To investigate effects of the injured rat pancreatic extract on in vitro trans-differentiation of bone marrow mesenchymal stem cells （BMSCs） into insulin-like cells. DESIGN, TIME AND SETTING： A cytology in vitro experiment was performed at the Institute of Biotechnology, Academy of Military Medical Sciences of Chinese PLA from June 2005 to March 2007. MATERIALS： Six clean male Sprague-Dawley rats were obtained from the Animal Experimental Center of Academy of Military Medical Sciences. Activin A, Retinoic Acid and trypsin inhibitor were bought from Sigma, USA. Streptozotocin was purchased from Sigma company. METHODS： Streptozotocin was intraperitoneally infused into rats. Two days later, pancreatic tissues were obtained when blood glucose exceeded 10 mmol/L. Homogenate was prepared in phosphate buffer saline containing pancreatic protein inhibitor. Supernatant was obtained after twice centrifuge, which was injured pancreatic extract. BMSCs were isolated from rats by density gradient centrifugation. The passage cells were randomly divided into 4 groups when 70%-80% passage cells were confluent. BMSCs in the control group were incubated in low-glucose DMEM containing 2% fetal bovine serum for 11 days. BMSCs in the Activin A and Retinoic Acid （AR） group were separately treated with Activin A and Retinoic Acid each for 24 hours, and with basic fibroblast growth factor, nicotinic amide, nicotinic amide＋exendin each for 3 days. BMSCs in the Activin A, Retinoic Acid and injured pancreatic extract （AR＋E） group were treated with injured pancreatic extract on the basis of former groups. BMSCs in the injured pancreatic extract （E） group were only incubated with injured pancreatic extract for 11 days. MAIN OUTCOME MEASURES： Phenotypes of cells were examined by irnmunocytochemistry and reverse transcription-polymerase chain reaction （RT-PCR）. Insulin secretion levels were determined by enzyme labeled immunosorbent assay （ELISA）. RESULTS： More insulin-positive cells existed in AR and AR＋E groups, and insulin-like structure was bigger and more in the AR＋E group compared with the AR group. There were a few insulin-positive cells and insulin-like structure in the E group. Insulin-1 mRNA expression existed in the experimental groups. Insulin secretion was detected in supernatant. Insulin secretion levels were greater in the AR＋E group compared with the AR group, and secretion levels were the lowest in the E group （P 〈 0.05）. Each index was negative in the control group. CONCLUSION： On the basis of previous study ofActivin A and Retinoic Acid and other maturation factors, the injured pancreatic extract can increase the efficiency of BMSCs differentiation into insulin-like cells, resulting in elevating differentiation efficiency.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第43期8458-8462,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
军队十一五计划项目(06Q086)~~
