腺病毒载体介导PDX-1在骨髓间充质干细胞中的表达

为研究PDX-1基因在骨髓间充质干细胞中的表达情况及生物学功能的发挥，构建了含PDX-1基因的重组腺病毒载体. 酶切PDX-1基因并连入穿梭质粒pAdTrack-CMV.用电穿孔法使穿梭质粒pAdTrack-CMV-PDX-1与病毒骨架质粒pAdEasy-1在大肠杆菌BJ5183中同源重组.利用脂质体介导重组腺病毒载体转染293细胞，包装出完整的腺病毒.分离、培养、扩增骨髓间充质干细胞.用重组腺病毒感染间充质干细胞.用荧光显微镜、RT-PCR、免疫荧光染色等方法检测PDX-1、胰岛素基因及蛋白质的表达，用放射免疫分析法检测转基因细胞分泌胰岛素情况.结果表明:通过测序、PCR、酶切等鉴定PDX-1基因已正确插入穿梭质粒中，并与病毒骨架质粒重组.重组腺病毒滴度为6.3×10⁷ PFU/ml.通过荧光显微镜观察证实重组腺病毒可高效感染骨髓间充质干细胞，经RT-PCR、免疫荧光染色证实转染pAd-PDX-1后培养7天的细胞中有PDX-1及胰岛素基因的表达.这些转基因的细胞向胞外分泌的胰岛素量为(15.21±3.50) mIU/L.

The Expression of PDX-1 Gene in Mesenchymal Stem Cells Transduced by Adenovirus Vector

To construct recombinant adenovirus vector contained human pancreatic duodenal homeobox-1 (PDX-1) so as to study the expression and effect of PDX-1 gene in bone marrow mesenchymal stem cells, human PDX-1 gene was ligated into shuttle vector pAdTrack-CMV. Homologous recombination was performed in BJ5183 bacteria by cotransforming linearized shuttle plasmid with adenovirus backbone plasmid pAdEasy-1.The recombinant plasmid was packaged and amplified in 293 cells.Mesenchymal stem cells(MSCs) were isolated from healthy adult bone marrow.The adenovirus was transfected into MSCs. The recombinant adenovirus vector has been successfully constructed according to the results of sequencing,PCR and enzyme digestion identification.The expression of PDX-1 and insulin in transfected MSCs at 7 days was detected by RT-PCR and immunocytochemistry. The value of insulin secretion was (15.21±3.50 )mIU/L from the transfected cells at 7days.These results showed that PDX-1 gene modified MSCs could be the beta cell replacement for those diabetes patients who need insulin-secreting cells.