Archive | 2019
Establishment of bone marrow mesenchymal stem cells stably expressing microRNA-23b mediated by lentivirus
Abstract
Objective \nTo establish a stable cell line which expresses microRNA-23b (named micro23b-BMSCs) using mice bone marrow mesenchymal stem cells (BMSCs) and lentivirus packaging system. \n \n \nMethods \nPrimary BMSCs were isolated from the femur and tibia of C57BL/6 mice.BMSCs were able to maintain the unique spiral growth state of stem cells and the potential for self-renewal after several passages.Lentiviral packaging was carried out using the plasmid pEZX-MR03 with the pre-miR23b gene, and the pMDLg/pRRE, pRSV Rev, pLP/VSVG.BMSCs were infected after collecting the virus solution.EGFP fluorescent label was observed under confocal microscopy.The infected mesenchymal stem cells were sieved with puromycin (2.5 μg/mL). \n \n \nResults \nBMSCs, which stably expressing miR-23b, were successfully established.RNA from these stem cells was extracted for reverse transcription and real-time fluorescent quantitative PCR(qPCR). The results of qPCR showed that the expression of miR23b in the overexpressed BMSCs was increased by 100 times compared with the control BMSCs(t=4.572, P<0.05). \n \n \nConclusion \nThe lentiviral packaging method can successfully integrate miR-23b into the genome of stem cells, which stably express miR-23b.This genetic modification approach provides a new combined approach to stem cell transplantation for disease. \n \n \nKey words: \nBone marrow mesenchymal stem cells (BMSCs);\xa0Lentiviral packaging system;\xa0MiR-23b