慢病毒载体介导miR-195低表达调控BMMSCs成骨分化及血管生成

doi:10.3870/j.issn.1672-8009.2026.05.006

医学分子生物学杂志 ›› 2026, Vol. 23 ›› Issue (5): 523-530.doi: 10.3870/j.issn.1672-8009.2026.05.006

慢病毒载体介导miR-195低表达调控BMMSCs成骨分化及血管生成

丁刘闯, 裴培, 姬晓炜

新疆医科大学第一附属医院口腔修复种植科乌鲁木齐市, 830013

收稿日期:2025-12-12 出版日期:2026-09-30 发布日期:2026-09-30
通讯作者: 姬晓炜（E-mail：31891275@qq.com）
基金资助:
“青年科研起航”基金青年项目（No.2024YFY-QKQN-86）

Effect of Lentiviral Vector-Mediated Knockdown of miR-195 on Osteogenic and Angiogenic Differentiation of BMMSCs

DING Liuchuang, PEI Pei, JI Xiaowei

Department of Prosthodontics and Implantology, First Affiliated Hospital of Xinjiang Medical University, Urumqi, 830013, China

Received:2025-12-12 Online:2026-09-30 Published:2026-09-30
Contact: JI Xiaowei （E-mail：31891275@qq.com ）
Supported by:
“Youth Research Start-up” Fund for Young Scholars（No.2024YFY-QKQN-86）

摘要/Abstract

摘要： 目的探讨慢病毒载体介导微小RNA-195（miR-195）低表达对骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMMSCs）成骨分化与成血管分化的调控作用及可能机制。方法将人BMMSCs分为空白对照组、inhibitor-NC组（慢病毒转染阴性对照NC序列）、miR-195-inhibitor组（慢病毒转染miR-195抑制序列）。采用RNA荧光原位杂交检测miR-195表达定位,CCK-8法检测增殖活力;碱性磷酸酶（alkaline phosphatase,ALP）染色、茜素红染色及Runt相关转录因子2（Runt-related transcription factor 2,Runx2）免疫荧光评估成骨分化;血管生成拟态实验结合qRT-PCR检测血管内皮生长因子（vascular endothelial growth factor,VEGF）、ALP、Runx2、VEGF受体2（VEGF receptor 2,VEGFR2）、促血管生成素（angiopoietin-1,Ang-1）及Wnt/β-catenin通路相关分子Wnt3a、Wnt10b、β-连环蛋白（β-catenin）、成骨细胞特异性转录因子（Osterix）的mRNA水平,蛋白质印迹检测上述通路及成骨相关蛋白表达。结果与对照组相比,miR-195-inhibitor组miR-195表达显著下调（P_均<0.05）,增殖活力、ALP活性（4.15±0.52 vs 1.00±0.15）、茜素红阳性率（64.67 %±7.78 % vs 31.86 %±2.24 %）、Runx2荧光强度（852.38±122.70 vs 121.31±12.16 RFU）、管状结构长度（218.60±24.75 vs 49.80±5.25 μm/视野）,VEGF、ALP、Runx2、VEGFR2、Ang-1、Wnt3a、Wnt10b、β-catenin、Osterix mRNA水平及Wnt3a、Wnt10b、β-catenin、Osterix蛋白水平均显著上调（P_均<0.05）,而与空白对照组比较,inhibitor-NC组的上述指标差异均无统计学意义（P>0.05）。结论慢病毒载体介导的miR-195低表达通过激活Wnt/β-catenin信号通路,协同促进BMMSCs成骨与成血管分化。

关键词: 慢病毒载体, 微小RNA-195, 骨髓间充质干细胞, 成骨分化, 成血管分化

Abstract: Objective To investigate the regulatory effect and potential mechanism of lentiviral vector-mediated microRNA-195 （miR-195） downregulation on osteogenic and angiogenic differentiation of bone marrow mesenchymal stem cells （BMMSCs）. Methods Human BMMSCs were divided into three groups： blank control group, inhibitor-negative control （NC） group （transfected with lentiviral NC sequence）, and miR-195-inhibitor group （transfected with lentiviral miR-195 inhibitor sequence）. The expression and localization of miR-195 were detected by RNA fluorescence in situ hybridization. Cell proliferation was measured by CCK-8 assay. Osteogenic differentiation was evaluated by alkaline phosphatase （ALP） staining, alizarin red staining, and Runt-related transcription factor 2 （Runx2） immunofluorescence. Tube formation assay was performed for angiogenesis assessment. The mRNA levels of vascular endothelial growth factor （VEGF）, ALP, Runx2, VEGF receptor 2 （VEGFR2）, angiopoietin-1 （Ang-1）, and Wnt/β-catenin pathway-related molecules （Wnt3a, Wnt10b, β-catenin, Osterix） were determined by qRT-PCR. The protein expression of the above pathway and osteogenesis-related molecules was detected by Western blotting. Results Compared with the control groups, the miR-195-inhibitor group showed significantly decreased miR-195 expression （all P<0.05）, and markedly increased cell proliferation, ALP activity （4.15±0.52 vs 1.00±0.15）, alizarin red positive rate （64.67%±7.78% vs 31.86%±2.24%）, Runx2 fluorescence intensity （852.38±122.70 vs 121.31±12.16 RFU）, and tubular structure length （218.60±24.75 vs 49.80±5.25 μm/field）. The mRNA levels of VEGF, ALP, Runx2, VEGFR2, Ang-1, Wnt3a, Wnt10b, β-catenin, and Osterix, as well as the protein levels of Wnt3a, Wnt10b, β-catenin, and Osterix were all significantly upregulated （all P<0.05）. There were no significant differences in the above indices between the inhibitor-NC group and the blank control group （P>0.05）. Conclusion Lentiviral-mediated miR-195 downregulation synergistically promotes osteogenic and angiogenic differentiation of BMMSCs via activating the Wnt/β-catenin signaling pathway.

Key words: lentivirus, microRNA-195, bone marrow mesenchymal stem cell, osteogenic differentiation, angiogenic differentiation

中图分类号:

R329.2

丁刘闯, 裴培, 姬晓炜. 慢病毒载体介导miR-195低表达调控BMMSCs成骨分化及血管生成[J]. 医学分子生物学杂志, 2026, 23(5): 523-530.

DING Liuchuang, PEI Pei, JI Xiaowei. Effect of Lentiviral Vector-Mediated Knockdown of miR-195 on Osteogenic and Angiogenic Differentiation of BMMSCs[J]. Journal of Medical Molecular Biology, 2026, 23(5): 523-530.

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慢病毒载体介导miR-195低表达调控BMMSCs成骨分化及血管生成

Effect of Lentiviral Vector-Mediated Knockdown of miR-195 on Osteogenic and Angiogenic Differentiation of BMMSCs

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