摘要
目的:研究胰腺十二指肠同源框-1(pancreatic duodenal homeobox-1,PDX-1)基因在骨髓间质干细胞(mesenchymal stem cells,MSCs)体外分化中的作用。方法:构建含有PDX-1基因的真核表达载体,转染介质Superfect介导重组载体转染MSCs,G418筛选阳性细胞后对转染组(PDX-1+MSCs)和未转染组(PDX-1-MSCs)均体外进行诱导分化,流式细胞仪检测胰岛素阳性细胞的比例,胰岛素ELISA试剂盒测定转染组诱导后细胞的胰岛素分泌功能。结果:限制性酶切分析和序列测定证实成功构建了含有PDX-1基因的真核表达载体,荧光显微镜观察证实Superfect高效介导重组载体转染MSCs;流式细胞仪发现PDX-1+MSCs分化为胰岛素分泌细胞的数量较PDX-1-MSCs明显增多(阳性率分别为28.23%±2.56%和7.08%±2.69%),25mmol高浓度葡萄糖刺激的胰岛素分泌量(115.29±2.56μU/mL)较5mmol低浓度葡萄糖刺激的分泌量明显增高(56.61±4.82μU/mL)。结论:PDX-1能增强大鼠MSCs体外分化为功能性胰岛素分泌细胞的能力,对开展胰岛移植治疗1型糖尿病有重要价值。
Objective： To investigate the possible effects of pancreatic duodenal homeobox- 1 （PDX- 1）expression in transdifferentiation of bone marrow mesenchymal stem cells（MSCs）in vitro. Methods： The eukaryotic expression vector containing PDX- 1 was constructed. The vector was transfected into MSCs using Superfect, G418 was used to select the positive cells. PDX- 1 ＋MSCs and PDX- 1-MSCs were induced to transdifferentiat ion in vitro, the proportion of insulin- producing cells was detected by flow cytometry. Insulin secretion function of PDX- 1＋MSCs was measured by ELISA. Results： Restricted enzyme analysis and sequencing showed that interest gene segment was consistent with that in Gene Bank. Recombination vector was effective transformed into MSCs demonstrated by fluorescence microscope, insulin- producing cells from PDX- 1 ＋MSCs were increased （28.23% ±2.56% and 7.08% ±2.69% respectively）, insulin secretion of PDX- 1 ＋MSCs challenged by glucose in 25 mmol exceeded that in 5 mmol （115.29±2.56 μU/mL and 56.61±4.82 μU/mL respectively）. Conclusion： PDX- 1 can promote adult rat MSCs to transdifferentiate into insulin- secreting cells in vitro.
出处
《中国现代普通外科进展》
CAS
2009年第1期12-17,共6页
Chinese Journal of Current Advances in General Surgery
关键词
间质干细胞
胰腺十二指肠同源框-1
干细胞移植
糖尿病
1型
Mesenchymal stem cells
Pancreatic duodenal homeobox- 1
Stem cell transplanta-tion
Diabetes mellitus,type 1
