EGCG/CeO2负载纳米颗粒在炎症微环境下对SCAPs的作用研究

doi:10.13591/j.cnki.kqyx.2025.07.009

摘要/Abstract

摘要：

目的探讨负载表没食子儿茶素-3-没食子酸酯(epigallocatechin-3-gallate,EGCG)及二氧化铈(cerium dioxide,CeO₂)的复合纳米颗粒E/Ce@MCSNs,在炎症微环境下对人根尖乳头干细胞(apical papilla stem cells,SCAPs)成牙/成骨向分化及巨噬细胞极化的影响。方法制备E/Ce@MCSNs并对其进行表征;CCK-8法检测不同浓度E/Ce@MCSNs对SCAPs及RAW 264.7细胞活力的影响;检测E/Ce@MCSNs的抗氧化模拟酶活性。模拟体外炎症微环境,采用DCFH-DA探针荧光法检测E/Ce@MCSNs对细胞内活性氧的清除能力;通过碱性磷酸酶(alkaline phosphatase,ALP)染色及活性检测、茜素红染色及半定量分析和实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,RT-qPCR)检测牙本质涎磷蛋白(dentin sialoprotein,DSPP)、ALP、核心转录因子-2(runt-related transcription factor 2,Runx-2)、Ⅰ型胶原(type Ⅰ collagen,COL-Ⅰ)、骨钙素(osteopontin,OPN)等成牙/成骨相关分化相关基因表达情况,评估E/Ce@MCSNs在该环境下对SCAPs成牙/成骨向分化的影响;采用RT-qPCR检测E/Ce@MCSNs对RAW264.7细胞肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)、白细胞介素(interleukin,IL)-1β、IL-6、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、转化生长因子-β(transforming growth factor-beta,TGF-β)和IL-10表达影响,并通过ELISA检测细胞培养上清TNF-α和IL-6浓度。结果当浓度≤100 μg/mL时,E/Ce@MCSNs具有良好的生物相容性;E/Ce@MCSNs具有优异的氧化物清除活性,可高效清除细胞内活性氧(P<0.05);E/Ce@MCSNs可显著增强炎症微环境中SCAPs的ALP活性(P<0.001)、促进钙结节形成(P<0.001),上调成牙/成骨相关基因(DSPP、ALP、Runx-2、COL-Ⅰ、OPN)的表达(P<0.05);E/Ce@MCSNs处理后,RAW264.7促炎因子(TNF-α、IL-1β、IL-6、iNOS)表达显著减少(P<0.01),抗炎因子(TGF-β、IL-10)表达显著增加(P<0.001),培养上清中TNF-α和IL-6水平显著降低(P<0.001)。结论 E/Ce@MCSNs通过清除过量活性氧,在根尖周炎中发挥抗氧化抗炎作用,促进SCAPs成牙/成骨向分化。

关键词: 活性氧, 抗氧化, 根尖乳头干细胞, 抗炎, 成骨分化

Abstract:

Objective To investigate the effects of composite nanoparticles E/Ce@MCSNs loaded with epigallocatechin-3-gallate (EGCG) and cerium dioxide (CeO₂) on the odonto/osteogenic differentiation of human stem cells from the apical papilla (SCAPs) and macrophage polarization under inflammatory conditions. Methods E/Ce@MCSNs were synthesized and characterized. Cell viability of SCAPs and RAW 264.7 cells treated with varying concentrations of E/Ce@MCSNs was assessed via CCK-8 assay. The antioxidant enzyme-mimetic activity of E/Ce@MCSNs was evaluated. Under simulated inflammatory conditions, intracellular reactive oxygen species (ROS) scavenging capacity was measured via DCFH-DA fluorescent probe. Alkaline phosphatase (ALP) staining/activity, alizarin red staining/semi-quantitative analysis, and RT-qPCR were performed to detect odonto/osteogenic differentiation markers, including dentin sialoprotein (DSPP), ALP, runt-related transcription factor 2 (Runx-2), type Ⅰ collagen (COL-Ⅰ), and osteopontin (OPN) in SCAPs. The effects of E/Ce@MCSNs on the odontogenic/osteogenic differentiation of SCAPs under this condition were evaluated. RT-qPCR were used to analyze cytokine expression (TNF-α, IL-1β, IL-6, iNOS, TGF-β, IL-10) and secreted TNF-α/IL-6 levels in RAW264.7 cells. The concentrations of TNF-α and IL-6 in cell culture supernatants were measured by ELISA. Results E/Ce@MCSNs exhibited excellent biocompatibility at concentrations ≤100 μg/mL. They demonstrated potent ROS-scavenging activity (P<0.05) and significantly enhanced ALP activity (P<0.001), promoted calcium nodule formation (P<0.001), and upregulated odonto/osteogenic gene expression (DSPP, ALP, Runx-2, COL-Ⅰ, OPN) in SCAPs under inflammatory conditions (P<0.05). In RAW264.7 cells, E/Ce@MCSNs downregulated pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, iNOS) (P<0.01) and upregulated anti-inflammatory factors (TGF-β, IL-10)(P<0.001), while reducing TNF-α and IL-6 secretion (P<0.001). Conclusion E/Ce@MCSNs exert antioxidant and anti-inflammatory effects in apical periodontitis by scavenging excessive ROS, thereby promoting odonto/osteogenic differentiation of SCAPs.

Key words: reactive oxygen species, antioxidant, apical papilla stem cells, anti-inflammatory, osteogenic differentiation

中图分类号:

R781.3

吴雨婷, 戴明睿, 冷迪雅, 朱婷婷, 吴滔, 吴大明. EGCG/CeO₂负载纳米颗粒在炎症微环境下对SCAPs的作用研究[J]. 口腔医学, 2025, 45(7): 529-539.

WU Yuting, DAI Mingrui, LENG Diya, ZHU Tingting, WU Tao, WU Daming. Study on the promotion of osteo/odontogenic differentiation on SCAPs and anti-inflammatory effects of EGCG/CeO₂-loaded nanoparticles in an inflammatory microenvironment[J]. Stomatology, 2025, 45(7): 529-539.

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基因	引物序列(5'-3')
ALP	F:GACCCTTGACCCCCACAAT R:GCTCGTACTGCATGTCCCCT
Runx-2	F:GGAGTGGACGAGGCAAGAGTTT R:AGCTTCTGTCTGTGCCTTCTGG
COL-Ⅰ	F:AAAGATGGACTCAACGGTCTC R:CATCGTGAGCCTTCTCTTGAG
DSPP	F:TTAAATGCCAGTGGAACCAT R:ATTCCCTTCTCCCTTGTGAC
OPN	F:GATGGCCTTGTATGCACCATTC R:GCAGACCTGACATCCAGTACC
GAPDH	F:CGCTCTCTGCTCCTCCTGTT R:CCATGGTGTCTGAGCGATGT

基因	引物序列(5'-3')
iNOS	F:GTTCTCAGCCCAACAATACAAGA R:GTGGACGGGTCGATGTCAC
IL-1β	F:AATCTCGCAGCAGCACATCAAC R:TGTTCATCTCGGAGCCTGTAGTG
IL-6	F:TTCCATCCAGTTGCCTTCTTG R:TCATTTCCACGATTTCCCAGAG
TNF-α	F:ACTGAACTTCGGGGTGATCG R:ATTCCCTTCTCCCTTGTGAC
TGF-β	F:CCACCTGCAAGACCATCGAC R:CTGGCGAGCCTTAGTTTGGAC
IL-10	F:CTTACTGACTGGCATGAGGATCA R:CCATGGTGTCTGAGCGATGT
β-actin	F:GCAGCTCTAGGAGCATGTGG R:AGCCACCAATCCACACAG

组别	S_BET/(m²/g)	V_p/(cm³/g)	D_p/nm
MCSNs	119.079 0	0.053 073	14.429 7
E/Ce@MCSNs	37.473 2	0.024 617	16.172 0

EGCG/CeO₂负载纳米颗粒在炎症微环境下对SCAPs的作用研究

Study on the promotion of osteo/odontogenic differentiation on SCAPs and anti-inflammatory effects of EGCG/CeO₂-loaded nanoparticles in an inflammatory microenvironment

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