miR-34a对人牙周膜干细胞增殖和成骨分化的影响

周锟; 刘亚丽; 李自良; 钱丽萍; 冉丽权; 任娅岚

doi:10.12259/j.issn.2095-610X.S20250403

miR-34a对人牙周膜干细胞增殖和成骨分化的影响

doi: 10.12259/j.issn.2095-610X.S20250403

周锟^{1, 2},
刘亚丽³,
李自良⁴,
钱丽萍⁴,
冉丽权⁴,
任娅岚^{2, 4, ,}

1.
昆明医科大学口腔医学院，云南昆明　650106
2.
云南省口腔医学重点实验室，云南昆明　650500
3.
昆明医科大学口腔医院正畸科，云南昆明　650106
4.
前兴路门诊部，云南昆明　650228

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（202301AY070001-010）；云南省口腔疾病临床医学研究中心青年科研基金（2022QN006）；云南省大学生创新创业训练计划基金（2023JXD061）；昆明医科大学大学生创新性实验基金（2022JXD196，2024CYD457）

详细信息

作者简介:
周锟（2003～），男，重庆人，在读本科生

通讯作者:
任娅岚，E-mail：670312366@qq.com

中图分类号: R329.2
计量
- 文章访问数: 259
- HTML全文浏览量: 119
- PDF下载量: 20
- 被引次数: 0
出版历程
- 收稿日期: 2024-12-24
- 网络出版日期: 2025-04-05
- 刊出日期: 2025-04-25

Effect of miR-34a on Proliferation and Osteogenic Differentiation of Human Periodontal Stem Cells

Kun ZHOU^{1, 2},
Yali LIU³,
Ziliang LI⁴,
Liping QIAN⁴,
Liquan RAN⁴,
Yalan REN^{2, 4
, ,}

1.
School of Stomatology，Kunming Medical University，Kunming Yunnan 650106
2.
Yunnan Provincial Key Laboratory of Stomatology，Kunming Yunnan 650500
3.
Department of Orthodontics，Hospital of Stomatology，Kunming Medical University，Kunming Yunnan 650106
4.
Qianxing Road Clinic，Hospital of Stomatology，Kunming Medical University，Kunming Yunnan 650228，China

摘要

摘要: 目的探讨miR-34a对人牙周膜干细胞增殖和成骨分化的影响。方法收集20颗因正畸治疗需要拔除的健康牙齿，体外分离培养人牙周膜干细胞（hPDLSCs），构建miR-34a的模拟物转染至hPDLSCs中，实验分组为mimics组（miR-34a过表达组）、mimics-NC组（空载组）。实时荧光定量PCR（qRT-PCR）法检验转染效率、CCK-8检测hPDLSCs在转染后的增殖能力。诱导转染miR-34a的hPDLSCs成骨分化，收集成骨分化后第0 d、14 d hPDLSCs，qRT-PCR法检测成骨标志Runt相关转录因子2（Runx2）的表达水平、Western blot法检测Runx2相关蛋白的表达水平、茜素红染色显示矿化结节形成情况。结果 mimics组的miR-34a表达水平显著高于mimics-NC组（P < 0.05）。第1～5天，mimics组、mimics-NC组的增殖率差异无统计学意义（P > 0.05）；第5～11天，mimics组增殖率明显低于mimics-NC组，差异有统计学意义（P < 0.05）。转染miR-34a的hPDLSCs经过成骨诱导后，在第0天和第14天 mimics组Runx2的mRNA表达水平均高于mimics-NC组（P < 0.05），mimics组Runx2蛋白表达水平也高于mimics-NC组（P < 0.05），成骨诱导14 d后 mimics组矿化结节多于mimics-NC组。结论在体外条件下，miR-34a抑制hPDLSCs的增殖活性，促进人牙周膜干细胞的成骨分化。
- miR-34a /
- 人牙周膜干细胞 /
- 增殖 /
- 成骨
Abstract: Objective To investigate the effects of miR-34a on proliferation and osteogenic differentiation of human periodontal stem cells. Methods Twenty healthy teeth that needed to be extracted for orthodontic treatment were collected. Human periodontal stem cells （hPDLSCs） were isolated and cultured in vitro, and miR-34a mimetics were constructed and transfected into hPDLSCs. The experimental groups were subsequently categorized into the mimics group （miR-34a overexpression group） and the mimics-NC group （control group without load）. The transfection efficiency was assessed using real-time fluorescence quantitative PCR （qRT-PCR）, while CCK-8 assays were used to evaluate the proliferation capacity of hPDLSCs post-transfection. Osteogenic differentiation of miR-34a-transfected hPDLSCs was induced, with samples being collected at day 0 and day 14 after the osteogenic induction. The expression level of Runx2-associated transcription factor 2 （Runx2） was quantified via qRT-PCR, protein levels of Runx2-associated proteins were analyzed through Western blot, and mineralized nodule formation was examined using alizarin red staining. Results The expression level of miR-34a in the mimics group was significantly higher than that in the mimics-NC group （P < 0.05）. There was no significant difference in the value-added rate between the mimics group and the mimics-NC group on days 1～5 （P > 0.05）, and the value-added rate between the mimics group and the mimics-NC group was significantly lower than that between the mimics-NC group and the mimics-NC group on days 5～11, and the difference was statistically significant. After the osteogenic induction, the mRNA expression level of Runx2 in the mimics group was higher than that in the mimics-NC group （P < 0.05）, and the expression level of Runx2 protein in the mimics group was also higher than that in the mimics-NC group （P < 0.05）, and there were more mineralized nodules in the mimics group than in the mimics-NC group after 14 days of osteogenic induction. Conclusion Under in vitro conditions, miR-34a inhibits the proliferative activity of hPDLSCs and promotes the osteogenic differentiation of human periodontal ligament stem cells.
- MicroRNA-34a /
- Human periodontal ligament stem cells /
- Proliferation /
- Osteogenesis

HTML全文

图 1 显微镜下hPDLSCs形态

A：原代细胞培养7 d（×40）；B：P1代细胞（×100）。

Figure 1. Morphology of hPDLSCs under microscope

下载: 全尺寸图片幻灯片

图 2 miR-34a转染效率测定

A：hPDLSCs转染6 h后荧光显微镜图（×40）；a：明视野；b：荧光视野；B：转染48 h后，hPDLSCs中miR-34a的表达（n = 3，^****P < 0.000 1）。

Figure 2. Transfection efficiency of miR-34a

下载: 全尺寸图片幻灯片

图 3 hPDLSCs增殖率

CCK-8法检测miR-34a对hPDLSCs不同处理组增殖率的影响（n = 5，^****P < 0.000 1，^*P < 0.05）。

Figure 3. Proliferation rate of hPDLSCs

下载: 全尺寸图片幻灯片

图 4 miR-34a促进hPDLSCs中Runx2 mRNA表达

A: 成骨诱导0 d Runx2 mRNA表达量；B: 成骨诱导14 d Runx2 mRNA表达量（n = 3，^****P < 0.000 1，^**P < 0.01）。

Figure 4. miR-34a promotes Runx2 mRNA expression in hPDLSCs

下载: 全尺寸图片幻灯片

图 5 miR-34a促进hPDLSCs中Runx2 蛋白表达

A: 成骨诱导后hPDLSCs中Runx2蛋白表达电泳图；B: 成骨诱导0 d Runx2蛋白表达量；C：成骨诱导14 d Runx2蛋白表达量（n = 3，^**P < 0.01，^*P < 0.05）。

Figure 5. miR-34a promotes Runx2 protein expression in hPDLSCs

下载: 全尺寸图片幻灯片

图 6 茜素红染色镜下观（A）和直条图（B）

A：成骨诱导14 d后显微镜下矿化结节（×100）；a：mimics组；b：mimics-NC组；B：成骨诱导14 d后矿化结节的相对含量（n = 3，^****P < 0.000 1）。

Figure 6. Microscopic view of alizarin red staining （A） and bar plot （B）

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primers sequences

基因	引物序列
miR-34a	Forward 5' -ACTGGTACTCAGACAACGAGAT-3'	Reverse 5' -ACGTCAATGTCCCTGATGTTATG-3'
U6	Forward 5' -AAAGCAAATCATCGGACGACC-3'	Reverse 5' -GTACAACACATTGTTTCCTCGGA-3'
Runx2	Forward 5' -CCGCCTCAGTGATTTAGGGC-3'	Reverse 5' -GGGTCTGTAATCTGACTCTGTCC-3'
β-actin	Forward 5' -TTGTCGCCCTTTTCTACTTTGCC-3'	Reverse 5' -CAATGTCCAGCCCATGATGGTTC-3'

下载: 导出CSV

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