鼠李糖乳杆菌胞外囊泡对MC3T3-E1细胞生物学特性的影响

doi:10.3969/j.issn.1674-8115.2026.09.004

上海交通大学学报（医学版） ›› 2026, Vol. 46 ›› Issue (9): 1188-1198.doi: 10.3969/j.issn.1674-8115.2026.09.004

• 论著 · 基础研究 • 上一篇

鼠李糖乳杆菌胞外囊泡对MC3T3-E1细胞生物学特性的影响

叶之韵¹, 周亦凝¹, 陈慧文¹, 何智妍², 周薇², 宋忠臣¹()

^1.上海交通大学医学院附属第九人民医院牙周病科，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011
^2.上海交通大学医学院附属第九人民医院口腔微生态与系统性疾病实验室，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200125

收稿日期:2025-09-23 接受日期:2026-04-16 出版日期:2026-09-28 发布日期:2026-09-28
通讯作者: 宋忠臣，主任医师，教授，博士；电子信箱：szhongchen@sina.com。
基金资助:
国家自然科学基金(82270974);上海交通大学医学院附属第九人民医院“交叉基金”(JYJC202225);上海交通大学医学院生物材料与再生医学研究院联合攻关项目(2022LHB04)

Effects of extracellular vesicles derived from Lactobacillus rhamnosus GG on the biological properties of MC3T3-E1 cells

Ye Zhiyun¹, Zhou Yining¹, Chen Huiwen¹, He Zhiyan², Zhou Wei², Song Zhongchen¹()

^1.Department of Periodontology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology, Shanghai 200011, China
^2.Laboratory of Oral Microbiota and Systemic Disease, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology, Shanghai 200125, China

Received:2025-09-23 Accepted:2026-04-16 Online:2026-09-28 Published:2026-09-28
Contact: Song Zhongchen, E-mail: szhongchen@sina.com.
Supported by:
National Natural Science Foundation of China(82270974);Cross-Disciplinary Research Fund of Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine(JYJC202225);Biomaterials and Regenerative Medicine Institute Cooperative Research Project of Shanghai Jiao Tong University School of Medicine(2022LHB04)

摘要/Abstract

摘要：

目的·观察鼠李糖乳杆菌胞外囊泡（extracellular vesicles derived from Lactobacillus rhamnosus GG，L.GG-EVs）对小鼠胚胎成骨细胞前体细胞（mouse embryonic osteoblast precursor cell line，MC3T3-E1）增殖、迁移与成骨分化的影响。方法·通过超速离心法分离提取L.GG-EVs后，使用透射电子显微镜（transmission electron microscope，TEM）观察L.GG-EVs形态，采用ZETAView纳米粒径追踪仪对L.GG-EVs的粒径分布进行检测，并经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）后使用考马斯亮蓝R-250染色观察L.GG-EVs的蛋白表达情况。采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）法检测共培养1、3、5、7 d后L.GG-EVs（12.5、25、50、100 μg/mL）对MC3T3-E1活力的影响。采用划痕愈合实验检测共培养6、24 h后L.GG-EVs（50、100 μg/mL）对MC3T3-E1迁移能力的影响。采用碱性磷酸酶（alkaline phosphatase，ALP）染色检测共培养7 d后L.GG-EVs（50、100 μg/mL）对MC3T3-E1中ALP表达的影响。采用茜素红S染色检测共培养21 d后L.GG-EVs（50、100 μg/mL）对MC3T3-E1钙结节形成的影响。采用实时荧光定量PCR（quantitative real-time PCR，qPCR）检测共培养7 d后L.GG-EVs（50、100 μg/mL）对MC3T3-E1成骨基因Runt相关转录因子2（Runt-related transcription factor 2，Runx2）、骨桥蛋白（osteopontin，Opn）和骨形态发生蛋白2（bone morphogenetic protein 2，Bmp2）mRNA表达的影响。构建大鼠牙槽骨缺损模型，分为手术组（SUR组）、单纯水凝胶［甲基丙烯酰胺基明胶（gelatin methacryloyl，GelMA）］组以及负载L.GG-EVs的水凝胶组（GelMA-L.GG-EVs组），采用Micro-CT检测大鼠牙槽骨再生情况。结果·L.GG-EVs呈双层膜类球形；直径为20~500 nm，呈正态分布，平均直径约为161 nm；其在相对分子质量42 000处有蛋白表达条带。CCK-8法的结果显示，与CON组相比，共培养3、5、7 d后L.GG-EVs（50、100 μg/mL）可显著促进MC3T3-E1增殖（均P<0.05）。划痕愈合实验的结果显示，与Ctrl组相比，L.GG-EVs（50、100 μg/mL）可加速MC3T3-E1的迁移（均P<0.05）。MC3T3-E1经共培养7 d后，L.GG-EVs（50、100 μg/mL）可使ALP染色显著加深（均P<0.05）。MC3T3-E1经共培养21 d后，L.GG-EVs（50、100 μg/mL）可使钙结节形成显著增加（均P=0.001）。共培养7 d后，50 μg/mL L.GG-EVs可上调MC3T3-E1的Opn和Bmp2 mRNA表达（均P<0.05），100 μg/mL L.GG-EVs可上调Runx2、Opn和Bmp2 mRNA表达（均P<0.05）。在大鼠牙槽骨缺损模型中，L.GG-EVs显著促进了牙槽骨再生（均P<0.05）。结论·L.GG-EVs可促进MC3T3-E1增殖、迁移与成骨分化，具有引导骨组织再生的潜力。

关键词: 鼠李糖乳杆菌, 胞外囊泡, 小鼠胚胎成骨细胞前体细胞, 增殖, 迁移, 成骨分化

Abstract:

Objective ·To observe the effects of extracellular vesicles derived from Lactobacillus rhamnosus GG (L.GG-EVs) on the proliferation, migration, and osteogenic differentiation of mouse embryonic osteoblast precursor cell line (MC3T3-E1). Methods ·After L.GG-EVs were extracted by ultracentrifugation, the morphology of L.GG-EVs was observed by transmission electron microscope (TEM), and the particle size distribution of L.GG-EVs was measured by ZETAView nanoparticle size tracker. After SDS-PAGE, Coomassie brilliant blue R-250 staining was used to observe the protein expression of L.GG-EVs. The effect of L.GG-EVs(12.5, 25, 50, and 100 μg/mL) on the viability of MC3T3-E1 cells after 1, 3, 5, and 7 d of co-culture was evaluated by a cell counting kit-8 (CCK-8) assay. The wound-healing assay was used to detect the effect of L.GG-EVs (50 and 100 μg/mL) on the migration ability of MC3T3-E1 cells after 6 and 24 h of co-culture. Alkaline phosphatase (ALP) staining was used to detect the effect of L.GG-EVs (50 and 100 μg/mL) on the ALP expression in MC3T3-E1 cells following 7 d of co-culture. Alizarin red S staining was used to detect the effect of L.GG-EVs (50 and 100 μg/mL) on calcium nodule formation in MC3T3-E1 cells after 21 d of co-culture. Quantitative real-time PCR (qPCR) was used to detect the mRNA expression of osteogenic genes, including Runt-related transcription factor 2 (Runx2), osteopontin (Opn), and bone morphogenetic protein 2 (Bmp2), in MC3T3-E1 cells after 7 d of co-culture with L.GG-EVs (50 and 100 μg/mL). A rat alveolar bone defect model was constructed and divided into the surgical group (SUR group), the pure hydrogel group [gelatin methacryloyl (GelMA) group], and the hydrogel group loaded with L.GG-EVs (GelMA-L.GG-EVs group). The alveolar bone regeneration was detected by Micro-CT. Results ·L.GG-EVs were spherical, double-layered membrane vesicles, with diameters ranging from 20 to 500 nm and an average diameter of approximately 161 nm, showing a normal distribution. A protein band was observed at a relative molecular mass of 42 000. CCK-8 assay showed that compared with the CON group, L.GG-EVs at 50 and 100 μg/mL significantly promoted the proliferation of MC3T3-E1 cells after co-culture for 3, 5, and 7 d (all P<0.05). The wound-healing assay indicated that L.GG-EVs (50 and 100 μg/mL) accelerated the migration of MC3T3-E1 cells compared with the Ctrl group (all P<0.05). After 7 d of co-culture, L.GG-EVs (50 and 100 μg/mL) markedly intensified ALP staining in MC3T3-E1 cells (both P<0.05). After 21 d of co-culture, L.GG-EVs (50 and 100 μg/mL) significantly increased calcium nodule formation in MC3T3-E1 cells (both P=0.001). After 7 d of co-culture, 50 μg/mL L.GG-EVs up-regulated the expression of Opn and Bmp2 mRNA (both P<0.05), and 100 μg/mL L.GG-EVs up-regulated the expression of Runx2, Opn, and Bmp2 mRNA (all P<0.05). In the rat alveolar bone defect model, L.GG-EVs significantly promoted alveolar bone regeneration (all P<0.05). Conclusion ·L.GG-EVs can promote the proliferation, migration, and osteogenic differentiation of MC3T3-E1 cells, and have the potential to facilitate bone tissue regeneration.

Key words: Lactobacillus rhamnosus GG (L.GG), extracellular vesicle (EV), mouse embryonic osteoblast precursor cell line (MC3T3-E1), proliferation, migration, osteogenic differentiation

中图分类号:

R781.4

叶之韵, 周亦凝, 陈慧文, 何智妍, 周薇, 宋忠臣. 鼠李糖乳杆菌胞外囊泡对MC3T3-E1细胞生物学特性的影响[J]. 上海交通大学学报（医学版）, 2026, 46(9): 1188-1198.

Ye Zhiyun, Zhou Yining, Chen Huiwen, He Zhiyan, Zhou Wei, Song Zhongchen. Effects of extracellular vesicles derived from Lactobacillus rhamnosus GG on the biological properties of MC3T3-E1 cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026,(9): 1188-1198.

图/表 8

表1 qPCR引物序列

Tab 1 Primer sequences for qPCR

Gene	Forward sequence (5′→3′)	Reverse sequence (5′→3′)
Runx2	CCGGGAATGATGAGAACTA	ACCGTCCACTGTCACTTT
Opn	AGCAAGAAACTCTTCCAAGCAA	GTGAGATTCGTCAGATTCATCCG
Bmp2	ATGTGAGGATTAGCAGGTCTTTG	TGGTTAGTGGAGTTCAGGTGGT
β-actin	GTGACGTTGACATCCGTAAAGA	GCCGGACTCATCGTACTC

图1 L.GG-EVs的表征Note： A. TEM images of L.GG-EVs (indicated by the yellow arrows). Magnification: ×5 000. B. Diameter distribution of L.GG-EVs. REL—relativity. C. SDS-PAGE analysis of L.GG-EVs.

Fig 1 Characterization of L.GG-EVs

图2 共培养1 d（A）、3 d（B）、5 d（C）、7 d（D）后不同浓度L.GG-EVs对MC3T3-E1增殖能力的影响

Fig 2 Effects of different concentrations of L.GG-EVs on the proliferation of MC3T3-E1 after 1 d (A), 3 d (B), 5 d (C), and 7 d (D) of co-culture

图3 共培养后不同时间点下50、100 μg/mL L.GG-EVs对MC3T3-E1迁移能力的影响Note： A. Effect of L.GG-EVs on the migration of MC3T3-E1 (×50). B. Percentage of scratch closure of MC3T3-E1 at 6 h and 24 h after scratching.

Fig 3 Effects of 50 and 100 μg/mL L.GG-EVs on the migration of MC3T3-E1 at different time points after co-culture

图4 共培养7 d后L.GG-EVs对MC3T3-E1中ALP表达的影响Note： A. Results of ALP staining (up: ×100). B. Statistical analysis of the integrated density of ALP staining in MC3T3-E1.

Fig 4 Effects of L.GG-EVs on the expression of ALP in MC3T3-E1 after 7 d of co-culture

图5 共培养21 d后L.GG-EVs对MC3T3-E1钙结节形成的影响Note： A. Results of alizarin red S staining (up: ×100). B. Statistical analysis of the formation of calcium nodules in MC3T3-E1.

Fig 5 Effects of L.GG-EVs on calcium nodule formation in MC3T3-E1 after 21 d of co-culture

图6 共培养7 d后L.GG-EVs对MC3T3-E1成骨标志物表达的影响Note： A. Relative expression of Runx2 mRNA. B.Relative expression of Opn mRNA. C. Relative expression of Bmp2 mRNA.

Fig 6 Effects of L.GG-EVs on the expression of osteogenic markers in MC3T3-E1 after 7 d of co-culture

图7 大鼠牙槽骨缺损模型中L.GG-EVs对牙槽骨再生的影响Note： A. Micro-CT images of alveolar bones. B. Statistical analysis of bone parameters.

Fig 7 Effects of L.GG-EVs on alveolar bone regeneration in a rat alveolar bone defect model

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鼠李糖乳杆菌胞外囊泡对MC3T3-E1细胞生物学特性的影响

Effects of extracellular vesicles derived from Lactobacillus rhamnosus GG on the biological properties of MC3T3-E1 cells

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