慢病毒介导的稳定表达miR-23b的骨髓间充质干细胞株的建立

Establishment of bone marrow mesenchymal stem cells stably expressing microRNA-23b mediated by lentivirus

目的通过慢病毒包装系统感染小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),建立能够稳定、高效表达microRNA-23b(miR-23b)的细胞株miR23b-BMSCs。方法从C57BL/6小鼠股骨及胫骨分离出来的原代BMSCs,经过多次传代培养,依然能保持干细胞特有的螺旋生长状态以及自我更新的潜能。利用带有pre-miR23b基因的质粒pEZX-MR03与pMDLg/pRRE、pRSV Rev、pLP/VSVG进行慢病毒包装,收集病毒液感染BMSCs,显微镜下观察带有eGFP荧光标记的间充质干细胞,利用嘌呤霉素(2.5μg/mL)进行药筛。结果成功筛选出稳定表达miR-23b的BMSCs,提取干细胞内RNA进行逆转录、实时荧光定量PCR(real-time fluorescent quantitative PCR,qPCR)进行表达鉴定。qPCR结果显示,过表达BMSCs组miR-23b的表达量较对照组BMSCs提高100倍(t=4.572,P<0.05)。结论利用慢病毒包装可以成功地将miR-23b整合到干细胞的基因组中,得到稳定表达miR-23b的小鼠BMSCs细胞株。这种基因改造的方法为干细胞移植治疗疾病提供了一种新的联合方法与治疗思路。

Objective To establish a stable cell line which expresses microRNA-23b(named micro23b-BMSCs)using mice bone marrow mesenchymal stem cells(BMSCs)and lentivirus packaging system.Methods Primary 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(2.5μg/mL).Results BMSCs,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).Conclusion The 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.