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miR-130b-3p促进人牙源性iPSCs重编程的作用

谭小兵 张敏 郭宇 杜琳玲 戴青原

谭小兵, 张敏, 郭宇, 杜琳玲, 戴青原. miR-130b-3p促进人牙源性iPSCs重编程的作用[J]. 昆明医科大学学报, 2022, 43(12): 18-22. doi: 10.12259/j.issn.2095-610X.S20221204
引用本文: 谭小兵, 张敏, 郭宇, 杜琳玲, 戴青原. miR-130b-3p促进人牙源性iPSCs重编程的作用[J]. 昆明医科大学学报, 2022, 43(12): 18-22. doi: 10.12259/j.issn.2095-610X.S20221204
Xiaobing TAN, Min ZHANG, Yu GUO, Linling DU, Qingyuan DAI. Effect of miR-130b-3p on the Reprogramming of Human Dental Origin iPSCs[J]. Journal of Kunming Medical University, 2022, 43(12): 18-22. doi: 10.12259/j.issn.2095-610X.S20221204
Citation: Xiaobing TAN, Min ZHANG, Yu GUO, Linling DU, Qingyuan DAI. Effect of miR-130b-3p on the Reprogramming of Human Dental Origin iPSCs[J]. Journal of Kunming Medical University, 2022, 43(12): 18-22. doi: 10.12259/j.issn.2095-610X.S20221204

miR-130b-3p促进人牙源性iPSCs重编程的作用

doi: 10.12259/j.issn.2095-610X.S20221204
基金项目: 云南省科技厅-昆明医科大学应用基础研究联合专项基金资助项目[2019FE001(-113)];云南省高层次卫生计生技术人才培养经费基金资助项目(D-2018045)
详细信息
    作者简介:

    谭小兵(1974~),男,山西运城人,医学硕士,副主任医师,主要从事牙体牙髓病的研究工作

    通讯作者:

    戴青原,E-mail:dqy0823@163.com

  • 中图分类号: R781

Effect of miR-130b-3p on the Reprogramming of Human Dental Origin iPSCs

  • 摘要:   目的  研究hsa-miR-130b-3p对人DPSCs重编程为iPSCs的影响。  方法  设计合成LV3(H1/GFP&Puro)-hsa-miR-130b-3p-mimics质粒,Lipofectamine 3000将质粒转染到人DPSCs,荧光显微镜观察细胞,RT-qPCR验证转染效率。Sendai reprogramming kit将2组DPSCs重编程为iPSCs(实验组为miR-130b-3p-DPSCs,对照组为DPSCs),对比2组iPSCs克隆形态和重编程效率,RT-PCR检测Oct4、Nanog、KOS、Klf4、c-Myc的表达。  结果  hsa-miR-130b-3p过表达质粒转染48 h后在DPSCs内有明显表达,证实高效的转染效率(P < 0.01)。Sev重编程得到的2组iPSCs均呈现为扁平致密的圆形克隆,边缘清晰平滑,集落内细胞形态均一、核质比较大,RT-PCR结果表明2组细胞均可表达干细胞特异标志物Oct4和Nanog,同时外源性病毒SeV或转录因子KOS/Klf4/c-Myc不再表达。实验组的重编程效率高于对照组(分别为0.037%和0.018%,P < 0.05)。  结论  hsa-miR-130b-3p可明显促进人DPSCs重编程的效率,为iPSCs应用于牙髓再生治疗提供研究基础。
  • 图  1  miR-130b-3p-mimics转染DPSCs效率验证

    A:miR-130b-3p空载组;B:miR-130b-3p-mimics组;C:2组细胞miR-130b-3p表达的q-PCR检测。**P < 0.01。

    Figure  1.  Validation of DPSCs transfection efficiency by miR-130b-3p-mimics

    图  2  2组iPSCs克隆形态和特异性标记物的表达

    A:DPSCs细胞形态(×100);B:正常DPSCs-iPSCs克隆;C:miR-130b-3p-DPSCs-iPSCs克隆(标尺 = 100 μm);D:2组iPSCs特异性标记物的RT-PCR结果。

    Figure  2.  Morphology and specific markers expression of iPSCs in two groups

    表  1  RT-PCR特异性引物的序列

    Table  1.   Specific primer sequences of RT-PCR

    基因引物序列(5′-3′)
    Sev Forward GGATCACTAGGTGATATCGAGC
    Reverse ACCAGACAAGAGTTTAAGAGA
    Klf4 Forward TTCCTGCATGCCAGAGGAGCCC
    Reverse AATGTATCGAAGGTGCTCAA
    Myc Forward TAACTGACTAGCAGGCTTGTCG
    Reverse TCCACATACAGTCCTGGATGAT
    KOS Forward ATGCACCGCTACGACGTGAGGGC
    Reverse ACCTTGACAATCCTGATGTGG
    Oct4 Forward GAAGGTATTCAGCCAAACGAC
    Reverse GTTACAGAACCACACTCGGA
    Nanog Forward TGCAAATGTCTTCTGCTGAGAT
    Reverse GTTCAGGATGTTGGAGAGTTC
    Gapdh Forward GGAGCGAGATCCCTCCAAAAT
    Reverse GGCTGTTGTCATACTTCTCATGG
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  • 收稿日期:  2022-07-27
  • 网络出版日期:  2022-12-05
  • 刊出日期:  2022-12-25

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