摘要
目的:研究Conophylline(CnP)联合Betacellulin-δ4(BTCδ4)对胎猪导管源胰腺干细胞的体外诱导分化作用.方法:在培养基中加入CnP、BTCδ4等诱导因子诱导胎猪导管源胰腺干细胞分化,台盼蓝检测CnP对胰岛样细胞团(islet-like cell cluster,ICC)的毒性,ICC行ELISA、免疫组织化学检测,逆转录-多聚酶链反应(RT-PCR)检测胰岛素基因、PDX-1等在未诱导、诱导细胞中的表达,葡萄糖刺激实验检测ICC分泌胰岛素的功能.结果:用0.1g/LCnP诱导ICC不显示明显的细胞毒性.诱导培养4-6wk,ELISA和免疫组织化学显示CnP联合BTCδ4提高了胰岛素分泌细胞量和胰岛素含量;RT-PCR显示提高了PDX-1,neuroD/Beta2,胰岛素等在ICC中的表达.葡萄糖刺激实验结果显示,诱导后,ICC合成、分泌胰岛素的能力显著增强(P<0.05).结论:CnP联合BTCδ4促进诱导胎猪导管源胰腺干细胞分化为胰岛素分泌细胞,分化细胞表达β细胞表面标志并能分泌胰岛素.
AIM： To investigate the impact of alkaloid conophylline （CnP） in combination with betacellulin-84 （BTCδ4） on the differentiation of porcine fetal pancreatic duct stem cells in vitro.
METHODS： For inducing cell differentiation, porcine fetal pancreatic duct stem cells were cultured in Medium 199 （M199） with CnP and BTC34, alone or in combination. Then cell viability was assessed by the trypan blue dye exclusion assay. Enzyme-linked immunosorbent assay （ELISA） and immunohistochemistry were used to measure the content of insulin released by islet-like cell clusters （ICCs）. The expression of PDX-1, Neuro D/Beta2 and insulin mRNAs was detected by reverse transcription-polymerase chain reaction （RT-PCR）. Insulin release in response to glucose was assessed by glucose load test.
RESULTS： Conophylline at concentrations below 0.1 g/L showed no marked toxicity toward the ICCs. Either CnP or BTC54 weakly enhanced the content of insulin, while CnP in combination with BTC54 synergistically increased cellular insulin content. The expression of PDX-1, insulin and Neuro D/Beta2 mRNAs could be detected in untreated ICCs. After treatment with CnP in combination with BTCδ4, the ICCs exhibited a prompt response to 25.0 mmol/L glucose by increasing insulin secretion.
CONCLUSION： CnP in combination with BTC64 potentiated the differentiation of porcine pancre- atic duct stem cells in cluster cultures towards B-cells.
出处
《世界华人消化杂志》
CAS
北大核心
2011年第20期2109-2115,共7页
World Chinese Journal of Digestology
基金
黑龙江省自然科学基金资助项目
No.D2007-05~~
