过表达Miro1慢病毒载体的构建及Miro1高表达间充质干细胞稳定转染细胞株的建立与鉴定

Construction of Miro1 overexpression lentiviral vectors and establishment and identification of Miro1^(Hi)-BMSCs stable transfection cell line

背景线粒体转移是干细胞发挥免疫修复功能的重要机制之一,Miro1是线粒体转移过程的关键蛋白,但其动力学研究欠缺研究模型。目的构建过表达Miro1的间充质干细胞稳转细胞株,为线粒体动力学研究间充质干细胞的抗炎修复机制提供细胞模型。方法通过PCR扩增目的基因后进行Gibson反应、转化及Gateway等方法构建载体,并对载体进行酶切鉴定。慢病毒感染间充质干细胞后,使用嘌呤霉素进行药物筛选获得稳转细胞株。后续分为三组进行相关鉴定。(1)BMSC组:正常间充质干细胞;(2)Con-BMSC组:慢病毒空载体感染的间充质干细胞;(3)Miro^(Hi)-BMSC组:过表达Miro1的慢病毒载体感染的间充质干细胞。通过RT-qPCR、Western blot检测上述三组细胞Miro1的表达水平,并进行划痕试验、水平迁移实验、成骨及成脂诱导分化以鉴定干细胞特性。结果所构建的载体完成酶切鉴定,并成功获得过表达Miro1重组慢病毒载体pLV-EGFP:T2A:Puro-EF1A>mRhot1/HA。经m RNA和蛋白水平检测,Miro^(Hi)-BMSC组的Miro1表达高于BMSC组和Con-BMSC组(P<0.05),干细胞水平、垂直迁移能力三组间差异无统计学意义(P>0.05),三组干细胞均可成功进行成脂及成骨诱导分化。结论通过慢病毒载体成功构建Miro1Hi-BMSCs稳转细胞株,且该稳转株仍保留干细胞特性,可用于间充质干细胞调控线粒体转移的相关研究。

Background Mitochondrial transfer is one of the important mechanisms for stem cells to exert immune repair function.Miro1 is the key protein in the process of mitochondrial transfer,but its dynamics research lacks research models.Objective To establish a stable Miro1 protein overexpressed mesenchymal stem cell line,so as to provide a cell model for further study on the anti-inflammatory and repair mechanism of mesenchymal stem cells from the perspective of mitochondrial dynamics.Methods The amplification of the target gene by PCR was firstly performed,followed by Gibson reaction,transformation and highthroughput Gateway to construct the vector,which was further identified by the enzyme digestion.The stable transfected mesenchymal stem cell line was then acquired by lentivirus transfection and puromycin drug screening and subsequently divided into three groups for identification,which were BMSC group with normal mesenchymal stem cells,Con-BMSC group with empty lentiviral vector stably transformed mesenchymal stem cells,and Miro1Hi-BMSC group with Miro1 overexpression lentiviral vector stably transformed mesenchymal stem cells.Expression level of Miro1 of above groups was detected by RT-qPCR and Western blot.Original natures of stem cells were identified by scratch test,vertical migration test,osteogenic and adipogenic differentiation induction.Results Recombinant Miro1 overexpressed lentivirus vector pLV-EGFP:T2A:Puro-EF1A>mRhot1/HA was successfully confirmed by digestion identification.Compared with the groups of BMSC and Con-BMSC,the expression level of Miro1 in Miro1^(Hi)-BMSC group was significantly higher by mRNA and protein level detection(P<0.05),while there were no significant differences in the horizontal and vertical migration ability among the three groups(P>0.05).The stem cells in the three groups could successfully differentiate into adipogenic and osteogenic cells.Conclusion Miro1^(Hi)-BMSCs stable transfection cell line is established by lentivirus vector,additionally,it retains original nature,which can be utilized for the further research on mitochondrial donation regulated by mesenchymal stem cells.