慢病毒载体介导的脂肪干细胞miR-1重组表达系统的构建

Construction of miR-1 recombinant expression system in adipose-derived stem cells mediated by lentiviral vector

目的构建miR-1稳定过表达并且适于体内示踪的基因修饰大鼠脂肪干细胞(rASCs)模型.方法首先对体外培养的rASCs进行表型鉴定及成骨、成脂诱导分化;然后观察重组慢病毒转染rASCs过程中不同感染复数(MOI)及有无添加聚凝胺(polybrene)对报告基因萤火虫荧光素酶(FLuc)表达和活性的影响;最后采用qRT-PCR检测miR-1-3p和miR-1-5p的表达水平.结果通过观察FLuc介导酶促反应引起的生物发光筛选出最佳转染条件为MOI 60,且添加聚凝胺能增强慢病毒转染rASCs的效率;随后应用该条件转染rASCs构建miR-1稳定过表达模型,qRT-PCR结果显示miR-1-3p和miR-1-5p的表达水平分别升高了65.6倍和37.4倍(P<0.05),证实该模型构建成功.结论慢病毒载体能成功介导外源基因在rASCs中稳定过表达,并且报告基因FLuc催化底物产生的生物发光有利于进一步的体内示踪研究.总言之,本研究为基因修饰细胞模型的构建提供了方法学基础,并为心肌组织再生修复提供新的工程细胞株.

Objective To construct a genetically modified rat adipose-derived stem cells(rASCs)model that stably overexpresses miR-1 and suitable for in vivo tracking.Methods The rASCs cultured in vitro were phenotyped and subjected to osteogenic and adipogenic induction;then the multiplicity of infection(MOI)of recombinant lentivirus and the transfection efficiency with or without polybrene treatment were investigated by observing the expression and activity of repoter gene firefly luciferase(FLuc)in rASCs;finally,the expression levels of miR-1-3p and miR-1-5p were detected using qRT-PCR.Results The results of bioluminescence induced by FLuc-mediated enzymatic reaction showed that the optimal transfection condition was MOI 60,and polybrene treatment enhanced the efficiency of lentiviral transfecting with rASCs.Subsequently,the transfection condition was used to construct a miR-1 stable overexpression model in rASCs.The results of qRT-PCR demonstrated that the expression levels of miR-1-3p and miR-1-5p increased by 65.6 and 37.4 folds,respectively.These findings suggest that the miR-1 stable expression model was successfully constructed.Conclusion In summary,the lentiviral vector can successfully mediate the stable overexpression of foreign genes in rASCs,and the bioluminescence generated by the reporter gene FLuc catalyzing substrate may be useful for the further in vivo tracing studies,this study provides a methodological basis for the construction of genetically modified cell models,as well as new engineered cell lines for myocardial tissue regeneration and repair.