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北京大学学报（医学版） ›› 2026, Vol. 58 ›› Issue (1): 10-21. doi: 10.19723/j.issn.1671-167X.2026.01.002

• 论著 • 上一篇    下一篇

miR-488-5p促进大鼠骨髓间充质干细胞成神经、成骨分化及神经化骨再生

曾立婷1, 程凯远1,2, 刘中宁1, 李健1, 杨静文1,*(), 姜婷1,*()   

  1. 1. 北京大学口腔医学院·口腔医院修复科, 国家口腔医学中心, 国家口腔疾病临床医学研究中心, 口腔生物材料和数字诊疗装备国家工程研究中心, 北京 100081
    2. 天津市口腔医院修复一科, 南开大学医学院, 天津市口腔功能重建重点实验室, 天津 300041
  • 收稿日期:2025-10-10 出版日期:2026-02-18 发布日期:2025-12-10
  • 通讯作者: 杨静文, 姜婷
  • 基金资助:
    国家自然科学基金(82170928); 国家自然科学基金(82201022)

miR-488-5p promotes osteogenic and neurogenic differentiation of rat bone marrow mesenchymal stem cells and enhances neuralized bone regeneration

Liting ZENG1, Kaiyuan CHENG1,2, Zhongning LIU1, Jian LI1, Jingwen YANG1,*(), Ting JIANG1,*()   

  1. 1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, Beijing 100081, China
    2. Department Ⅰ of Prosthodontics, Tianjin Stomatological Hospital, School of Medicine, Nankai University, Tianjin Key Laboratory of Oral and Maxillofacial Function Reconstruction, Tianjin 300041, China
  • Received:2025-10-10 Online:2026-02-18 Published:2025-12-10
  • Contact: Jingwen YANG, Ting JIANG
  • Supported by:
    the National Natural Science Foundation of China(82170928); the National Natural Science Foundation of China(82201022)

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摘要：

目的: 探讨miR-488-5p促进大鼠骨髓间充质干细胞(rat bone marrow mesenchymal stem cells, rBMSCs)成神经或成骨分化的作用, 以及对神经化骨再生的影响。方法: 体外诱导rBMSCs向成神经或成骨向分化, 实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction, qRT-PCR)检测成神经或成骨分化过程中不同时间点(第0、2、4、7天)miR-488-5p的表达水平。建立miR-488-5p模拟物组、miR-488-5p抑制剂组以及各自的阴性对照组(negative control, NC)共四组, 分别探讨miR-488-5p对rBMSCs神经向分化及成骨向分化的影响。构建大鼠颅骨缺损模型(直径5 mm全层圆形临界骨缺损), 光固化甲基丙烯酰化明胶(gelatin methacryloyl, GelMA)负载rBMSCs形成以下四组并进行体内成骨实验: ①BLANK组: GelMA; ②BMSCs组: GelMA+rBMSCs; ③NC-BMSCs组: GelMA+转染miR-488-5p模拟物NC的rBMSCs; ④miR-488-5p-BMSCs组: GelMA+转染miR-488-5p模拟物的rBMSCs。术后4周和8周获取标本, 进行显微CT扫描及影像学骨参数分析, 并通过苏木精-伊红染色、Masson染色和神经特异性蛋白可溶性蛋白100(soluble protein-100, S100)组织免疫荧光染色, 评估缺损区神经化组织工程骨的生成效果。结果: 成功诱导rBMSCs向成神经或成骨向分化, 在rBMSCs成神经或成骨向分化过程中, miR-488-5p从第0天至第7天表达水平均显著升高。给予干预后, rBMSCs的模拟物组成神经相关基因及蛋白表达较其对照组增高, 抑制剂组则呈相反结果; 模拟物组的成骨相关基因及蛋白表达较其对照组增高, 碱性磷酸梅(alkaline phosphatase, ALP)染色及茜素红染色加深, ALP活性增强, 抑制剂组则呈相反结果。骨缺损动物模型结果显示, 术后4周和8周, BLANK组新生骨量最少, BMSCs组和NC-BMSCs组新生骨量居中且相近, miR-488-5p-BMSCs组新生骨量最多, 4周时miR-488-5p-BMSCs组骨矿物质密度[(0.63±0.05) g/cm3 vs. (0.51±0.03) g/cm3]、骨体积分数(33.17%±6.43% vs. 18.11%±1.52%)、骨表面积密度[(3.43±0.69) /mm vs. (2.46±0.20) /mm]及骨小梁数量[(0.92±0.21) /mm vs.(0.59±0.07) /mm]显著高于NC-BMSCs组, 8周时miR-488-5p-BMSCs组的骨矿物质密度[(0.80±0.04) g/cm3 vs. (0.68±0.04) g/cm3]、骨体积分数(56.69%±6.22% vs. 42.36%±3.86%)及新生骨周围被S100标记的神经细胞数量(46.33±4.04 vs. 26.00±3.61)显著高于NC-BMSCs组。结论: miR-488-5p同时具有促进rBMSCs成神经及成骨分化的作用, 并促进大鼠颅骨缺损神经化组织工程骨的形成。

关键词: miR-488-5p, 骨髓间充质干细胞, 成骨分化, 神经向分化, 神经-骨组织工程

Abstract:

Objective: To investigate the role of microRNA miR-488-5p, which showed increased expression after the disconnection of the inferior alveolar nerve, in promoting the osteogenic and neurogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs), as well as its effect on promoting the neuralized tissue engineered bone regeneration. Methods: rBMSCs were subjected to in vitro neural or osteogenic differentiation induction cultures. The expression levels of miR-488-5p at different time points (days 0, 2, 4, and 7) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). miR-488-5p overexpression or low expression in rBMSCs was achieved by transfection with miR-488-5p mimics or inhibitors. Four groups, the miR-488-5p mimics, the miR-488-5p inhibitor, and their respective negative controls (NC), were established to investigate the effects of miR-488-5p on the neural differentiation and osteogenic differentiation of rBMSCs.A 5 mm diameter, full-thickness circular critical bone defect was created in the rat calvaria. The rats were treated with light-cured gelatin methacryloyl (GelMA) seeded with rBMSCs. The rats were divided into four groups: ①BLANK group: GelMA; ②BMSCs group: GelMA + rBMSCs; ③NC-BMSCs group: GelMA + rBMSCs transfected with miR-488-5p mimics NC; and ④miR-488-5p-BMSCs group: GelMA + rBMSCs transfected with miR-488-5p mimics. Specimens were obtained 4 and 8 weeks after surgery, and micro-CT was performed to measure and analyze bone mineral density (BMD), bone volume/total volume (BV/TV), bone surface area/total volume (BS/TV) and trabecular number (Tb.N). The effects of neuralized tissue engineering bone formation in the defect area were assessed using Hematoxylin-Eosin (HE) staining, Masson staining, and tissue immunofluorescence staining of the nerve-specific protein soluble protein-100 (S100). Results: As rBMSCs progressed toward neural or osteogenic differentiation, miR-488-5p expression increased significantly from day 0 to day 7. Regarding neural differentiation, the mimics group showed increased expression of neural-related genes and proteins compared with the mimics NC group, while the opposite result was observed in the inhibitor group. As for osteogenic differentiation, the mimics group showed increased expression of osteogenic genes and proteins, more intense alkaline phosphatase (ALP) and alizarin red staining (ARS) staining, and enhanced ALP activity compared with the mimics NC group, while the opposite result was observed in the inhibitor group. 4 and 8 weeks after critical calvarial defect construction in rats, the BLANK group had the least amount of new bone formation, while the BMSCs group and the NC-BMSCs group had similar and intermediate amounts of new bone formation. The miR-488-5p-BMSCs group had the most new bone formation. At 4 weeks, the BMD [(0.63±0.05) g/cm3 vs. (0.51±0.03) g/cm3], BV/TV (33.17%±6.43% vs. 18.11%±1.52%), BS/TV [(3.43±0.69) /mm vs. (2.46±0.20) /mm], and Tb.N [(0.92±0.21) /mm vs.(0.59±0.07) /mm] in the miR-488-5p-BMSCs group were significantly higher than those in the NC-BMSCs group. At 8 weeks, the BMD [(0.80±0.04) g/cm3 vs. (0.68±0.04) g/cm3], BV/TV (56.69%±6.22% vs. 42.36%±3.86%), and the number of S100-labeled nerve cells around the new bone (46.33±4.04 vs. 26.00±3.61) in the miR-488-5p-BMSCs group was also significantly higher than that in the NC-BMSCs group. Conclusion: miR-488-5p promoted the osteogenic and neurogenic differentiation of rBMSCs and promoted the formation of neuralized tissue-engineered bone in rat calvarial defects.

Key words: miR-488-5p, Bone marrow mesenchymal stem cells, Osteogenic differentiation, Neurogenic differentiation, Neuro-bone tissue engineering

中图分类号: 

  • R68

表1

qRT-PCR引物序列"

Gene Forward primer (5′ to 3′) Reverse primer (5′ to 3′)
U6 CGCTTCGGCAGCACATATAC CGAATTTGCGTGTCATCCTT
miR-488-5p GGCACCCAGATAATGGCAC AGTGCAGGGTCCGAGGTATT
Gapdh AGGTCGGTGTGAACGGATTT GAACTTGCCGTGGGTAGAGT
*Map2 ACAGCAACAAGTGGTGAATCAG GGAGGATGGAGGAAGGTCTTG
*Ng2 TTCTCACACAGAGGAGCCC CACTCAAGCTCTGGCTGCT
*Tubb3 CAGATGCTGGCCATTCAGAGTAAG TGTTGCCGATGAAGGTGGAC
*Ngf TGCCAAGGACGCAGCTTTC TGAAGTTTAGTCCAGTGGGCTTCAG
*Nes TGAACAAGAGACCCAACAAACAC TTCCAAGAGGCTTCGGTAACT
*S100 CTGTCAAGAACCTGCTCCGA AGTGGGCATGGAACACATTGA
#Runx2 GCCTTCAAGGTTGTAGCCCT TGAACCTGGCCACTTGGTTT
#Alp CATGGTGAGTGACACGGACA CCATGACGTGGGGGATGTAG
#Ocn CCGTTTAGGGCATGTGTTGC TTTCGAGGCAGAGAGAGGGA
#Bmp2 CGGGAACAAATGCAGGAAGC AAGGACATTCCCCATGGCAG
#Col1a1 CCCCAGCCGCAAAGAGTCTA CAGCTGACTTCAGGGATGTCTTC
#Opn CCAGCCAAGGACCAACTACA AGTGTTTGCTGTAATGCGCC

图1

miR-488-5p促进rBMSCs神经向分化"

图2

miR-488-5p促进rBMSCs成骨向分化"

图3

大鼠颅骨缺损术后4周和8周micro-CT影像学分析"

图4

大鼠颅骨缺损术后4周和8周组织形态学分析"

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ISSN 1671-167X
CN 11-4691/R
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