大肠杆菌外膜囊泡依赖靶细胞内吞抑制大鼠胶质瘤细胞增殖

doi:10.12122/j.issn.1673-4254.2026.08.02

摘要/Abstract

摘要：

目的探讨大肠杆菌外膜囊泡（E. coli-OMVs）抑制胶质瘤增殖及其机制。方法采用 CCK-8 检测不同浓度 E. coli-OMVs 对C6胶质瘤细胞活力的影响，设置PBS对照组及2.5、5、10、20 μg/mL E. coli-OMVs组。设置PKH67-E. coli-OMVs组及2.5、5、10、20 μmol/L 氯丙嗪（CPZ）+PKH67-E. coli-OMVs组，观察CPZ对细胞摄取OMVs的影响。设置PBS对照组、10 μg/mL E. coli-OMVs 组及2.5、5、10 μmol/L CPZ+10 μg/mL E. coli-OMVs组，采用EdU掺入实验评价细胞增殖能力。基于上述结果，选择PBS对照组、10 μg/mL E. coli-OMVs组及10 μmol/L CPZ+10 μg/mL E. coli-OMVs组，采用qRT-PCR、Western blotting、免疫细胞化学和流式细胞术检测PCNA/Ki67表达及细胞周期分布。设置PBS对照组、10 μg/mL E. coli-OMVs组、CHIR99021组及CHIR99021+10 μg/mL E. coli-OMVs组，检测Wnt/β-catenin通路相关蛋白β-catenin、Cyclin D1和c-Myc的表达。建立SD大鼠原位胶质瘤模型，经鼻腔给予PBS或0.25、0.5、1.0 mg/kg E. coli-OMVs，动态监测肿瘤生长，并结合HE染色及PCNA、Ki67免疫组化评价其抗肿瘤作用。结果 2.5~10 μg/mL E. coli-OMVs可不同程度抑制C6细胞活力（P<0.05）。CPZ可减弱细胞对 OMVs的摄取，其中 20 μmol/L CPZ对细胞活力产生明显影响。2.5、5、10 μmol/L CPZ均可不同程度减弱OMVs的抗增殖作用，其中10 μmol/L CPZ干预效果最明显。与PBS组相比，10 μg/mL E. coli-OMVs可降低EdU阳性率（P<0.001），下调PCNA和 Ki67 的 mRNA（P<0.01）及蛋白表达（P<0.001），增加G0/G1期细胞比例并降低S期细胞比例（P<0.001）；与 E. coli-OMVs组相比，10 μmol/L CPZ共处理可显著回升PCNA mRNA（P<0.01）、PCNA蛋白（P<0.01）、Ki67 mRNA（P<0.05）及 Ki67 蛋白表达（P<0.001），G0/G1期细胞比例下降，S期细胞比例回升（P<0.001）。10 μg/mL E. coli-OMVs可下调β-catenin、Cyclin D1和c-Myc表达（P<0.001），而CHIR99021可部分恢复上述蛋白水平（P<0.001）。动物实验显示，0.25、0.5、1.0 mg/kg E. coli-OMVs鼻腔给药均可降低原位胶质瘤相对荧光信号，减缓肿瘤生长（P<0.001），并减少肿瘤组织中PCNA和Ki67的表达（P<0.001）。结论 E. coli-OMVs可抑制胶质瘤细胞增殖并延缓原位胶质瘤生长，其作用可能与肿瘤细胞对OMVs的内吞摄取、Wnt/β-catenin 信号通路抑制及细胞周期阻滞有关。

关键词: 大肠杆菌, 外膜囊泡, 胶质瘤细胞, 内吞作用, PCNA, Ki67

Abstract:

Objective To investigate the inhibitory effect of Escherichia coli outer membrane vesicles (E. coli-OMVs) on glioma proliferation and the underlying mechanism. Methods CCK-8 assay and EdU incorporation assay were used to determine the optimal concentrations of E. coli-OMVs and chlorpromazine (CPZ) for cell treatment. C6 glioma cells treated with 10 μg/mL E. coli-OMVs and 10 μmol/L CPZ were examined for PCNA and Ki67 expressions and cell cycle distribution using qRT-PCR, Western blotting, immunocytochemistry and flow cytometry. The expressions of β-catenin, cyclin D1, and c-Myc proteins in C6 cells were detected following treatment with OMVs in the presence or absence of CHIR99021. In a SD rat model bearing orthotopic glioma, the effects of intranasal PBS or OMVs (0.25, 0.5, 1.0 mg/kg) administration on tumor growth and PCNA and Ki67 expressions were observed using fluorescence imaging and immunohistochemistry. Results E. coli-OMVs within the concentrations of 2.5-10 μg/mL dose-dependently inhibited C6 cell viability, and such inhibitory effect was attenuated by 2.5, 5, and 10 μmol/L CPZ, which was the most effective at 10 μmol/L. The OMVs at 10 μg/mL significantly reduced EdU-positive cells, downregulated mRNA and protein expressions of PCNA and Ki67, and increased G0/G1-phase and decreased S-phase cell populations. These alterations were largely reversed by 10 μmol/L CPZ. Treatment with OMVs downregulated cellular expressions of β‑catenin, Cyclin D1, and c-Myc, which were partially restored by application of CHIR99021. In the tumor-bearing mice, intranasal OMVs administration dose-dependently decreased the relative fluorescence signals, slowed tumor growth, and reduced PCNA and Ki67 expressions. Conclusion E. coli-OMVs inhibit glioma cell proliferation and delay orthotopic tumor growth in rats likely via endocytic uptake of the OMVs to cause suppression of Wnt/β-catenin signaling and cell cycle arrest.

Key words: Escherichia coli, outer membrane vesicles, glioma cells, endocytosis, PCNA, Ki67

胡晨曦, 顾曦, 牛鹏程, 王祎婷, 方廖琼, 王智彪, 白晋. 大肠杆菌外膜囊泡依赖靶细胞内吞抑制大鼠胶质瘤细胞增殖[J]. 南方医科大学学报, 2026, 46(8): 1730-1742.

Chenxi HU, Xi GU, Pengcheng NIU, Yiting WANG, Liaoqiong FANG, Zhibiao WANG, Jin BAI. Escherichia coli outer membrane vesicles inhibit rat glioma proliferation through cell endocytosis[J]. Journal of Southern Medical University, 2026, 46(8): 1730-1742.

图/表 12

图1 E. coli-OMVs的提取与表征

Fig.1 Isolation and characterization of Escherichia coli outer membrane vesicles (E. coli-OMVs). A: Transmission electron microscopy of purified OMVs (Scale bar=200 nm). B: Size distribution of OMVs measured by NTA. C: Western blotting of OMV markers OmpA and OmpC.

图2 CCK-8分析E. coli-OMVs对胶质瘤细胞活力的影响

Fig.2 CCK-8 analysis of the effect of E. coli-OMVs on glioma cell viability. A: Glioma cell viability after 12-h co-culture with E. coli-OMVs. B: Glioma cell viability after 24-h co-culture with E. coli-OMVs. C: Glioma cell viability after 48-h co-culture with E. coli-OMVs (n=3). *P<0.05, **P<0.01, ***P<0.001 vs control group.

图3 CPZ对胶质瘤细胞摄取E. coli-OMVs的影响

Fig.3 Effect of CPZ on uptake of E. coli-OMVs by glioma cells. A: CCK-8 analysis of C6 cell viability after treatment with different concentrations of CPZ (n=3). B: Representative fluorescence images of C6 cells incubated with fluorescently labeled E. coli-OMVs after pretreatment with different concentrations of CPZ (Scale bar=75 μm). C: Representative fluorescence images of C6 cells incubated with fluorescently labeled E. coli-OMVs for 0 (control), 2 and 4 h (Scale bar=10 μm). D: Representative fluorescence images of C6 cells pretreated with 10 μmol/L CPZ and then incubated with fluorescently labeled E. coli-OMVs for 0, 2 and 4 h (Scale bar=10 μm). ***P<0.001 vs PBS.

图4 CPZ对E. coli-OMVs抑制胶质瘤细胞体外增殖的影响

Fig.4 CPZ attenuates inhibitory effect of E. coli OMVs on glioma cell proliferation in vitro. A-C: Representative EdU staining images after 12, 24, and 48 h treatment (Scale bar=50 μm). D-F: Quantification of EdU-positive cells after 12, 24 and 48 h treatment (n=3). ***P<0.001 vs control. #P<0.05,##P<0.01, ###P<0.001 vs E. coli OMVs.

图5 CPZ对E. coli-OMVs调控胶质瘤细胞PCNA与Ki67转录水平的影响

Fig.5 Effect of CPZ on PCNA and Ki67 transcriptional levels in E. coli-OMVs-treated glioma cells. A: qRT-PCR analysis of PCNA mRNA expression levels. B: qRT-PCR analysis of Ki67 mRNA expression levels (n=3). **P<0.01, ***P<0.001 vs PBS group; #P<0.05, ##P<0.01 vs E. coli-OMVs group.

图 6 CPZ对E. coli-OMVs调控胶质瘤细胞PCNA蛋白表达的影响

Fig.6 Effect of CPZ on PCNA protein expression in E. coli-OMVs-treated glioma cells. A: Western blots showing PCNA protein levels. B: Quantitative analysis (grayscale values) of the protein bands (n=3). ***P<0.001 vs PBS group; ##P<0.01 vsE. coli-OMVs group.

图7 CPZ对E. coli-OMVs调控胶质瘤细胞Ki67蛋白表达的影响

Fig.7 Effect of CPZ on Ki67 protein expression in E. coli-OMVs-treated glioma cells. A: Immunofluorescence images of Ki67 expression (Scale bar=50 μm). B: Quantitative analysis of Ki67-positive cells (n=3). ***P<0.001 vs PBS group; ##P<0.01 vs E. coli-OMVs group.

图8 CPZ 对 E. coli-OMVs 调控胶质瘤细胞周期分布的影响

Fig.8 Effect of CPZ on cell cycle distribution of E. coli-OMVs-treated glioma cells. A: Flow cytometry of C6 cell cycle distribution in PBS, E. coli-OMVs and CPZ+E. coli-OMVs groups. B: Quantitative analysis of the percentages of cells in G0/G1, S and G2/M phases (n=3). ***P<0.001 vs PBS group; ##P<0.01, ###P<0.001 vs E. coli-OMVs group.

图9 E. coli-OMVs 通过调控 Wnt/β-catenin 信号通路抑制胶质瘤细胞增殖相关蛋白表达

Fig.9 E. coli-OMVs inhibit expressions of glioma cell proliferation-related proteins by regulating the Wnt/β-catenin signaling pathway. A: Western blots of β‑catenin, cyclin D1 and c-Myc proteins in PBS, OMVs, CHIR99021 and CHIR99021+OMVs groups. B: Quantitative analysis of β-catenin, cyclin D1 and c-Myc protein expression levels. (n=3). ***P<0.001 vs PBS group; ###P<0.01 vs E. coli- OMVs group.

图10 E. coli-OMVs 鼻腔给药对大鼠原位胶质瘤生长的影响

Fig.10 Effect of intranasal administration of E. coli-OMVs on growth of orthotopic glioma in rats. A: In vivo bioluminescence images of orthotopic glioma-bearing rats on day 1, day 4, and day 7 after administration. B: Quantitative analysis of relative fluorescence intensity on day 4 (Day4/Day1). C: Quantitative analysis of relative fluorescence intensity on day 7 (Day7/Day1). D: Changes in body weight of rats in each group during treatment. OMVs: Outer membrane vesicles (n=3). **P<0.01, ***P<0.001 vs control group.

图11 E. coli-OMVs 鼻腔给药对大鼠原位胶质瘤组织形态的影响

Fig.11 Effect of intranasal administration of E. coli-OMVs on histopathological changes in orthotopic glioma tissues of the rats (Scale bar=20 μm).

图12 E. coli-OMVs 鼻腔给药对大鼠原位胶质瘤组织中增殖相关蛋白表达的影响

Fig.12 Effect of intranasal administration of E. coli-OMVs on expressions of proliferation-related proteins in orthotopic glioma tissues of the rats (Scale bar=20 μm). A: Immunohistochemical staining and quantitative analysis of PCNA expression in brain tumor tissues in each group. B: Immunohistochemical staining and quantitative analysis of Ki67 expression in brain tumor tissues in each group (n=3). ***P<0.001 vs PBS group.

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