细胞膜囊泡递送靶向肿瘤坏死因子-α的小干扰RNA对牙髓干细胞的抗炎作用

doi:10.19723/j.issn.1671-167X.2026.01.003

摘要/Abstract

摘要：

目的: 探索细胞膜囊泡(cell membrane vesicles, CMVs)作为广泛适用的基因沉默工具小干扰RNA(small interfering RNA, siRNA)递送平台的可行性, 并以脂多糖(lipopolysaccharide, LPS)诱导的人牙髓干细胞(dental pulp stem cells, DPSCs)炎症模型验证其应用效果。方法: 采用细胞松弛素B处理3T3细胞制备CMVs, 并通过共聚焦显微镜、动态光散射检测CMVs理化性质。利用洋地黄皂苷透化CMVs将靶向肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)的siRNA(siTNF-α)负载至CMVs中, 构建CMVs@siTNF-α。通过流式细胞术和共聚焦显微镜检测siRNA负载与胞内转运效率。以LPS(1 mg/L)刺激DPSCs建立炎症模型, 并与CMVs或CMVs@siTNF-α共培养。采用细胞活力检测试剂盒(cell counting kit-8, CCK-8)检测细胞毒性, 实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction, qRT-PCR)和酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)检测TNF-α、白介素(interleukin, IL)-1β和IL-6的表达与分泌。结果: CMVs呈球形, 由细胞膜及细胞质构成, 平均粒径为903 nm, 表面电位为-9.39 mV。流式细胞术结果显示, 负载荧光素酰胺(fluorescein amidite, FAM)标记siRNA(FAM-siRNA)后CMVs荧光强度升高; DPSCs加入CMVs@FAM-siTNF-α培养24 h后, 荧光信号增强。CCK-8检测结果显示CMVs及CMVs@siTNF-α对DPSCs无明显毒性。LPS处理显著上调DPSCs中TNF-α、IL-1β和IL-6的表达, 与LPS组相比, CMVs@siTNF-α下调TNF-α mRNA及其蛋白表达水平, 并抑制IL-1β和IL-6的转录与分泌。单独CMVs处理无明显差异。结论: CMVs可作为一种低毒性的siRNA递送平台, 在体外炎症模型中实现TNF-α沉默及下游炎症反应抑制, 具有进一步研究和应用的潜力。

关键词: 小干扰RNA, 细胞膜囊泡, 牙髓干细胞, 肿瘤坏死因子

Abstract:

Objective: To evaluate the feasibility of using cell membrane vesicles (CMVs) as a delivery system for small interfering RNA (siRNA) and to assess their performance in a lipopolysaccharide (LPS)-induced inflammatory model of human dental pulp stem cells (DPSCs). Methods: CMVs were generated from cytochalasin B-treated 3T3 cells and characterized for their physicochemical properties, including morphology, size distribution, and zeta potential, using confocal microscopy and dynamic light scattering. To construct CMVs@siTNF-α, saponin-mediated transient permeabilization was used to facilitate siRNA loading, after which encapsulation efficiency was evaluated by flow cytometry and confocal imaging. Intracellular uptake behaviors were examined using flow cytometry in DPSCs. LPS (1 mg/L) was employed to establish a robust in vitro inflammatory microenvironment. DPSCs were subsequently treated with CMVs or CMVs@siTNF-α, and cell viability was assessed via CCK-8 (cell counting kit-8). The expression and secretion of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme linked immunosorbent assay (ELISA) to evaluate both gene-silencing efficiency and downstream anti-inflammatory effects. Results: CMVs exhibited uniform spherical morphology with an average diameter of approximately 903 nm and a zeta potential of -9.39 mV, confirming successful vesicle formation. CMVs efficiently encapsulated FAM-labeled siRNA, as indicated by a pronounced fluorescence shift in flow cytometry and clear colocalization signals in confocal imaging. DPSCs cultured with CMVs@FAM-siTNF-α demonstrated increased intracellular fluorescence, reflecting efficient vesicle uptake and cytoplasmic siRNA release. Importantly, both CMVs and CMVs@siTNF-α displayed negligible cytotoxicity. LPS stimulation significantly elevated TNF-α, IL-1β, and IL-6 expression, validating the inflammatory model. CMVs alone did not affect cytokine levels, indicating biological inertness. In contrast, CMVs@siTNF-α significantly suppressed the LPS-induced upregulation of TNF-α at both mRNA and protein levels., demonstrating potent gene-silencing activity. Furthermore, suppression of TNF-α led to downstream attenuation of IL-1β and IL-6, with both transcription and secretion significantly decreased compared with the LPS group. These findings collectively confirmed that CMVs enabled efficient intracellular siRNA transport and effectively mitigate inflammatory responses in DPSCs. Conclusion: CMVs represent a biocompatible and effective siRNA delivery platform capable of achieving robust TNF-α knockdown and ameliorating inflammatory cytokine production in vitro, highlighting their potential for future nucleic acid-based anti-inflammatory therapies.

Key words: Small interfering RNA, Cell membrane vesicles, Dental pulp stem cells, Tumor necrosis factor

中图分类号:

高若凡, 马天宇, 王润楷, 殷雨辰, 李芮迪, 王丹丹, 夏斌. 细胞膜囊泡递送靶向肿瘤坏死因子-α的小干扰RNA对牙髓干细胞的抗炎作用[J]. 北京大学学报（医学版）, 2026, 58(1): 22-29.

Ruofan GAO, Tianyu MA, Runkai WANG, Yuchen YIN, Ruidi LI, Dandan WANG, Bin XIA. Anti-inflammatory effects of cell membrane vesicle-mediated delivery of small interfering RNA targeting tumor necrosis factor-α on dental pulp stem cells[J]. Journal of Peking University (Health Sciences), 2026, 58(1): 22-29.

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Gene	Forward primer (5′ to 3′)	Reverse primer (5′ to 3′)
TNF-α	AGCAAGGACAGCAGAGGA	GGGGAGAGAGGGTGGAG
IL-1β	TGTGCTGAATGTGGACTCA	ACAAAAGGGCTGGGGAT
IL-6	TGTGTGAAAGCAGCAAAGA	ACCAGGCAAGTCTCCTCA
GAPDH	GCCAACGTGTCAGTGGTG	AAGGTGGAGGAGTGGGTGT

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