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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

陈镜伊 赵玲萍 刘雯丽 习佳飞 梁志欣

陈镜伊, 赵玲萍, 刘雯丽, 习佳飞, 梁志欣. 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[J]. 解放军医学院学报, 2023, 44(4): 408-416. doi: 10.3969/j.issn.2095-5227.2023.04.015
引用本文: 陈镜伊, 赵玲萍, 刘雯丽, 习佳飞, 梁志欣. 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[J]. 解放军医学院学报, 2023, 44(4): 408-416. doi: 10.3969/j.issn.2095-5227.2023.04.015
CHEN Jingyi, ZHAO Lingping, LIU Wenli, XI Jiafei, LIANG Zhixin. Construction of Miro1 overexpression lentiviral vectors and establishment and identification of Miro1Hi-BMSCs stable transfection cell line[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL, 2023, 44(4): 408-416. doi: 10.3969/j.issn.2095-5227.2023.04.015
Citation: CHEN Jingyi, ZHAO Lingping, LIU Wenli, XI Jiafei, LIANG Zhixin. Construction of Miro1 overexpression lentiviral vectors and establishment and identification of Miro1Hi-BMSCs stable transfection cell line[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL, 2023, 44(4): 408-416. doi: 10.3969/j.issn.2095-5227.2023.04.015

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

doi: 10.3969/j.issn.2095-5227.2023.04.015
基金项目: 基础加强计划重点基础研究项目(2021-JCJQ-ZD-077-12)
详细信息
    作者简介:

    陈镜伊,女,在读硕士。Email: chenjingyi301@163.com

    通讯作者:

    梁志欣,男,博士,主任医师,教授。Email: liangzhixin301@163.com

  • 中图分类号: R329.2

Construction of Miro1 overexpression lentiviral vectors and establishment and identification of Miro1Hi-BMSCs stable transfection cell line

More Information
  • 摘要:   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) MiroHi-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mRNA和蛋白水平检测,MiroHi-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。

     

  • 图  1  过表达载体与对照空载体设计图谱

    A:pLV-EGFP:T2A:Puro-EF1A>mRhot1[NM_021536.8]/HA;B:pLV-EGFP:T2A:Puro-EF1A>mCherry

    Figure  1.  Gene mapping of the overexpression and control vectors

    A: pLV-EGFP:T2A:Puro-EF1A>mRhot1[NM_021536.8]/HA; B: pLV-EGFP:T2A:Puro-EF1A>mCherry

    图  2  终载体的酶切鉴定结果

    A:SnapGene软件查找酶切位点;B:限制性内切酶酶切产物琼脂糖凝胶电泳

    Figure  2.  Identification of the final vector by enzyme digestion

    A: Finding the digestion sites by the SnapGene software; B: Agarose gel electrophoresis map of the products from the enzyme digestion

    图  3  病毒载体感染HEK293T细胞后48 h荧光图(100×)

    A:质粒DNA转染HEK293T细胞包装病毒明场图;B:质粒DNA转染HEK293T细胞包装病毒EGFP荧光图;C:病毒感染HEK293T细胞明场图;D:病毒感染HEK293T细胞EGFP荧光图(MOI=5)

    Figure  3.  Photos of HEK293T cells taken by fluorescence microscopy after 48 h of transduction (100×)

    A: Plasmid DNA transfected virus packaged by HEK293T cells which observed through bright-field microscope; B: Plasmid DNA transfected virus packaged by HEK293T cells which observed through fluorescence microscopy; C: The virus transduced HEK293T cells which observed through bright - field microscope; D: The virus transduced HEK293T cells which observed through fluorescence microscopy (MOI=5)

    图  4  慢病毒载体转染BMSCs荧光图(MOI=40,转染11 d,药筛6 d,200×)

    A:对照慢病毒载体转染BMSCs明场图 ;B:对照慢病毒载体转染BMSCs mCherry荧光图;C:对照慢病毒载体转染BMSCs EGFP荧光图;D:Miro1 过表达载体转染BMSCs明场图;E:Miro1过表达载体转染BMSCs EGFP荧光图

    Figure  4.  Photos of BMSCs taken by fluorescence microscopy after transduction of lentiviral vectors (MOI=40, 11 days after the transduction, 6 days after screening test by puromycin, 200×)

    A: Control lentiviral vectors transduced BMSCs which observed through bright-field microscope; B: Control lentiviral vectors transduced BMSCs which observed through mCherry fluorescence microscopy; C: Control lentiviral vectors transduced BMSCs which observed through EGFP fluorescence microscopy; D: Miro1 protein overexpressed lentiviral vectors transduced BMSCs which observed through bright-field microscope; E: Miro1 protein overexpressed lentiviral vectors transduced BMSCs which observed through EGFP fluorescence microscopy

    图  5  BMSC、Con-BMSC及Miro1Hi-BMSC细胞株的Miro1表达水平鉴定

    A:蛋白水平Western blot结果;B:mRNA水平RT-qPCR结果

    Figure  5.  Identification of Miro1 expression levels among groups of BMSC, Con-BMSC and Miro1Hi-BMSC

    A: Western blot results of protein; B: RT-qPCR results of mRNA

    图  6  BMSC、Con-BMSC及Miro1Hi-BMSC细胞株的迁移能力鉴定

    A:水平迁移实验结果及统计分析(40×),P>0.05;B:垂直迁移实验结果及统计分析(100×),P>0.05

    Figure  6.  Migration capacity identification among groups of BMSC, Con-BMSC and Miro1Hi-BMSC

    A: Results of scratch test and statistical analysis (40×), P>0.05; B: Results of vertical migration test and statistical analysis (100×), P>0.05

    图  7  BMSC、Con-BMSC及Miro1Hi-BMSC细胞株的分化能力鉴定

    A:成骨诱导分化26 d (200×);B:成脂诱导分化20 d (400×)

    Figure  7.  Differentiative capacity identification among groups of BMSC, Con-BMSC and Miro1Hi- BMSC

    A: 26 days after osteogenesis induces differentiation (200×); B: 20 days after adipogenesis induces differentiation (400×)

    表  1  mRhot1引物序列

    Table  1.   Primer sequences of mRhot1

     mRNA引物序列
    mRhot1-FCAACTTTGTACAAAAAAGCAGGCTGCCACCATGCGTGCGGGCCGAGTG
    mRhot1-RTCAAGCGTAATCTGGAACATCGTATGGGTATCGCTGTTTCAGTAGTGCTCTGTAC
    下载: 导出CSV

    表  2  反应体系构成

    Table  2.   Composition of the reaction system

    组分体积/μL
    2⊆SYBR Green PCR MasterMix10.0
    引物0.3
    cDNA3.0
    去离子水6.7
    下载: 导出CSV
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出版历程
  • 收稿日期:  2022-11-16
  • 网络出版日期:  2023-04-06
  • 刊出日期:  2023-04-28

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