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

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

1.
云南省第一人民医院口腔医学中心/昆明理工大学附属医院，云南昆明　650032
2.
昆明医科大学第一附属医院心内科，云南昆明　650032

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
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出版历程
- 收稿日期: 2022-07-27
- 网络出版日期: 2022-12-05
- 刊出日期: 2022-12-25

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

1.
Center of Stomatology，The 1st People’s Hospital of Yunnan Province/The Affiliated Hospital of Kunming University of Science and Technology，Kunming Yunnan 650032
2.
Dept. of Cardiology，The 1st Affiliated Hospital of Kunming Medical University，Kunming Yunnan 650032，China

摘要

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。
- 牙髓干细胞 /
- 重编程 /
- 诱导性多能干细胞 /
- 微小RNAs /
- 过表达
Abstract: Objective To explore the effect of hsa-miR-130b-3p on the reprogramming of human dental iPSCs. Methods LV3 （H1/GFP&Puro）-hsa-miR-130b-3p-mimics plasmid was designed and synthesized. The plasmid was transduced into human DPSCs by Lipofectamine 3000 kit according to the manufacturer’s instructions. Fluorescence microscopy was used to observe the cells and transfection efficiency was verified by RT-qPCR. Sendai reprogramming kit was used to induce two groups of DPSCs （miR-130b-3p-DPSCs in the experimental group and DPSCs in the control group） into iPSCs. The morphology and reprogramming efficiency were compared and the expression of Oct4/Nanog/KOS/Klf4/c-Myc was detected by RT-PCR in two groups of iPSCs. Results hsa-miR-130b-3p overexpression plasmid was significantly expressed in DPSCs 48h after transfection, indicating high transfection efficiency （P < 0.01）. The two groups of iPSCs obtained by Sev reprogramming showed flat and dense round clones with clear and smooth edges, uniform cell morphology and large nucleolus in the colonies. RT-PCR results showed that cells in both groups could express the specific markers of stem cells Oct4 and Nanog. At the same time, exogenous virus SeV or transcription factors KOS/Klf4/c-Myc were no longer expressed. RT-PCR showed that cells in both groups all expressed specific markers Oct4 and Nanog, while SeV or KOS/Klf4/ C-MYC were not detected. The reprogramming efficiency of the experimental group was higher than that of the control group （0.037% and 0.018% respectively, P < 0.05）. Conclusion hsa-miR-130b-3p could promote the reprogramming efficiency of human DPSCs. Our research provides theoretical foundation for dental iPSCs application in dental pulp regenerative therapy.
- Dental pulp stem cells /
- Reprogramming /
- Induced pluripotent stem cells /
- microRNAs /
- Overexpression

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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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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
Sev	Reverse	ACCAGACAAGAGTTTAAGAGA
Klf4	Forward	TTCCTGCATGCCAGAGGAGCCC
Klf4	Reverse	AATGTATCGAAGGTGCTCAA
Myc	Forward	TAACTGACTAGCAGGCTTGTCG
Myc	Reverse	TCCACATACAGTCCTGGATGAT
KOS	Forward	ATGCACCGCTACGACGTGAGGGC
KOS	Reverse	ACCTTGACAATCCTGATGTGG
Oct4	Forward	GAAGGTATTCAGCCAAACGAC
Oct4	Reverse	GTTACAGAACCACACTCGGA
Nanog	Forward	TGCAAATGTCTTCTGCTGAGAT
Nanog	Reverse	GTTCAGGATGTTGGAGAGTTC
Gapdh	Forward	GGAGCGAGATCCCTCCAAAAT
Gapdh	Reverse	GGCTGTTGTCATACTTCTCATGG

下载: 导出CSV

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