Construction of retrovirus vector taking MDR1/ACBC1 and its transfection into human placenta derived mesenchymal stem cells
In the study, we used both the methods of perfusion and density gradient centrifugation to isolate and purify mesenchymal stem cells (MSCS) from placenta tissue, and constructed a retroviral vector with multiple drug resistant genes, and the green fluorescent protein (GFP) has been used as an indicative mark. The 293T cell was transfected by the retroviral vector PMX-flag-MDR1-GFP together with its peripheral membrane protein gene. After the infective and replication–defective retrovirus were acquired, we transfected them into human placenta-derived mesenchymal stem cells (HPMSCs). We successfully observed the expression of the reporter gene-GFP by using the green light fluorescence microscope and the p-glycoprotein (P-gp) expressed by exogenous gene MDR1 by Western Blotting. All these facts indicated that the retroviral vector PMX-flag-MDR1-GFP had successfully been transfected into HPMSCs and the exogenous gene multidrug resistance (MDR)1 was detected as normally expressed. The daunorubicin (DNR) pump experiment proved that P-gp of HPMSCs transfected with PMX-flag-MDR1-GFP was of biological activity. The result indicates that MDR1 retroviral vector can transfect the HPMSCs. Not only can the exogenous gene be expressed, but also the expression protein had the biological activity. The conclusion lays a solid foundation of the clinical application of MDR1 genetic therapy.
Keywords: Transfect, human placenta-derived mesenchymal stem cells, multidrug resistance (MDR)1 gene.