Li Jing, Zhu Li, Zhao Chun-Hua
Center of Excellence in Tissue Engineering, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2011 Dec;33(6):675-8.
To explore the feasibility of using a virus-free system in the induction of umbilical cord derived mesenchymal stem cells (UC-MSCs) into insulin-secreting cells.
MSCs were isolated from human umbilical cord and induced into insulin-secreting cells with a three-stage method. The mRNA expression levels of foxa2, sox17, pdx1, ngn3, pax4, insulin, and glut-2 were compared between induced and non-induced groups by RT-PCR in each stage. The distribution pattern of insulin and c-peptide were detected by immunofluorescence staining and observed by fluorescence microscopy. Insulin and c-peptide secretion and glucose responsiveness were detected by enzyme-linked immunosorbent assay (ELISA).
Transcription factors foxa2, sox17, pdx1, ngn3, pax4, insulin, and glut-2 were expressed in the induced cells. The mRNA expression levels of foxa2 and sox17 were significantly higher in the induced group than those in non-induced group in the first stage (all P < 0.05), pdx1, ngn3, and pax4 were significantly higher in the induced cells than those in non-induced cells in the second stage (all P < 0.05), and insulin and glut-2 expressions were significantly up-regulated in the induced group at last stage (all P < 0.05). Immunofluorescence staining showed that insulin and c-peptide were located in the cytoplasm of more than 90% of induced cells. ELISA showed that total intracellular insulin content of the induced cells contained up to (346.3 739 +/- 32.5 149) microU/ml, which was significantly higher than insulin in non-induced cells (17.69 +/- 1.46) microU/ml (P < 0.01). C-peptide content of the induced cells measured up to (195.10 +/- 8.88) pmol/L/h (P < 0.01), when exposed to 5.5 mmol/L glucose (P < 0.01). When stimulated with 22 mmol/L glucose, the c-peptide content of the induced cells increased to (340.99 +/- 7.91) pmol/L/h (P < 0.01 ).
The umbilical cord derived MSCs can be efficiently induced into insulin-secreting cells via a virus-free system.
探讨使用无病毒系统将脐带间充质干细胞（UC-MSCs）诱导分化为胰岛素分泌细胞的可行性。
从人脐带中分离间充质干细胞，采用三阶段法将其诱导为胰岛素分泌细胞。在每个阶段，通过逆转录聚合酶链反应（RT-PCR）比较诱导组和未诱导组中foxa2、sox17、pdx1、ngn3、pax4、胰岛素和glut-2的mRNA表达水平。通过免疫荧光染色检测胰岛素和C肽的分布模式，并通过荧光显微镜观察。采用酶联免疫吸附测定（ELISA）检测胰岛素和C肽分泌以及葡萄糖反应性。
诱导细胞中表达转录因子foxa2、sox17、pdx1、ngn3、pax4、胰岛素和glut-2。在第一阶段，诱导组中foxa2和sox17的mRNA表达水平显著高于未诱导组（均P<0.05）；在第二阶段，诱导细胞中pdx1、ngn3和pax4的表达水平显著高于未诱导细胞（均P<0.05）；在最后阶段，诱导组中胰岛素和glut-2的表达显著上调（均P<0.05）。免疫荧光染色显示，超过90%的诱导细胞的细胞质中存在胰岛素和C肽。ELISA显示，诱导细胞的细胞内总胰岛素含量高达（346.3739±32.5149）μU/ml，显著高于未诱导细胞中的胰岛素含量（17.69±1.46）μU/ml（P<0.01）。当暴露于5.5mmol/L葡萄糖时，诱导细胞的C肽含量高达（195.10±8.88）pmol/L/h（P<0.01）。当用22mmol/L葡萄糖刺激时，诱导细胞的C肽含量增加至（340.99±7.91）pmol/L/h（P<0.01）。
通过无病毒系统可将脐带间充质干细胞高效诱导为胰岛素分泌细胞。
