神经元素3基因沉默对脐源性胰岛前体细胞MafA表达的影响

目的 探讨神经元素3（Ngn3）基因在人脐带间充质干细胞（hUMSCs）向胰岛前体细胞分化过程中的作用以及对肌腱膜纤维肿瘤基因同系物A（MafA）表达的影响。方法 组织块法分离培养并鉴定hUMSCs；采用分阶段联合诱导法诱导hUMSCs向胰岛前体细胞分化，将其分为正常诱导组、基因沉默组、空病毒转染组，后两组在诱导第7天分别转染干扰病毒和空病毒，嘌呤霉素筛选后检测感染效果；21 d诱导结束后倒置相差显微镜和电子显微镜观察细胞形态变化；免疫细胞化学技术检测诱导后各组细胞胰岛素、胰高血糖素、Ngn3的表达；Real-time PCR、Western blotting检测Ngn3以及MafA的表达变化。结果 成功分离培养hUMSCs；分阶段联合诱导使正常诱导组和空病毒转染组细胞分化为胰岛前体细胞，Ngn3、胰岛素、胰高血糖素呈阳性表达，电子显微镜观察可见分泌颗粒；成功沉默了基因沉默组细胞的Ngn3基因，且该组细胞Ngn3、胰岛素、胰高血糖素呈阴性表达；Ngn3、MafA在基因沉默组明显下降（P<0.05）。结论 Ngn3基因沉默阻滞了hUMSCs向胰岛前体细胞的分化，并抑制了MafA的表达。

Effects of neurogenin 3 gene silencing on MafA expression in umbilical-derived pancreatic progenitor cells#br#

Objective To investigate the role of neurogenin 3 (Ngn3) gene in the differentiation of human umbilical cord mesenchymal stem cells (hUMSCs) into pancreatic progenitor cells and the effect on Mus musculus v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A(MafA) expression.Methods Isolated, cultured and identified the hUMSCs. Inducing the differentiation of hUMSCs into insulin-secreting cells in stages, and divided them into normal induction group, gene silencing group, and empty virus transfection group.The latter two groups were transfected with interfering virus and empty virus on the 7th day of induction.Detection of infection after puromycin screening was performed. After 21 days of induction,observed the changes of cell morphology by the inverted phase contrast microscope and electron microscope, then detecting the expression of insulin, glucagon and Ngn3 in each group of cells by immunocytochemical technique, detecting the changes of Ngn3 and MafA expression by Real-time PCR and Western blotting.Results The hUMSCs were successfully isolated and cultured.The cells in the normal induction group and the empty virus transfection group were differentiated into pancreatic progenitor cells by stage induction, and Ngn3, Insulin, and glucagon were positively expressed, and secreted particles were observed under electron microscope. Successfully silenced the Ngn3 gene of the gene silencing group of cells, and the cells of this group showed negative expression of Ngn3, insulin and glucagon.Ngn3 and MafA decreased significantly in the gene silencing group (P<0.05).Conclusion Ngn3 gene silencing blocked the differentiation of hUMSCs into insulin-secreting cells and suppressed the expression of MafA.