龈沟液中细胞外游离DNA与牙周临床指标及环磷酸鸟苷—磷酸腺苷合成酶—干扰素基因刺激因子信号通路相关分子的关联性分析

doi:10.7518/hxkq.2025.2024391

华西口腔医学杂志 ›› 2025, Vol. 43 ›› Issue (6): 808-818.doi: 10.7518/hxkq.2025.2024391

龈沟液中细胞外游离DNA与牙周临床指标及环磷酸鸟苷—磷酸腺苷合成酶—干扰素基因刺激因子信号通路相关分子的关联性分析

陈兰¹(), 朱轩智¹, 周婕妤¹, 李继遥², 赵蕾¹()

^1.口腔疾病防治全国重点实验室国家口腔医学中心国家口腔疾病临床医学研究中心四川大学华西口腔医院牙周病科，成都 610041
^2.口腔疾病防治全国重点实验室国家口腔医学中心国家口腔疾病临床医学研究中心四川大学华西口腔医院牙体牙髓病科，成都 610041

收稿日期:2024-10-23 修回日期:2025-09-23 出版日期:2025-12-01 发布日期:2025-11-27
通讯作者: 赵蕾 E-mail:2444713576@qq.com;zhaolei@scu.edu.cn
作者简介:陈兰，医师，博士，E-mail：2444713576@qq.com
基金资助:
国家自然科学基金(81970944);国家自然科学基金(81991501);国家自然科学基金(81991502);四川省自然科学基金(2023NSFSC0553)

Correlation analysis of cell-free DNA in gingival crevicular fluid with periodontal clinical indicators and cyclic guanosine phosphate-adenosine phosphate synthase-stimulator of interferon genes signaling pathway

Chen Lan¹(), Zhu Xuanzhi¹, Zhou Jieyu¹, Li Jiyao², Zhao Lei¹()

^1.State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Dept. of Periodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
^2.State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Dept. of Cariology and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China

Received:2024-10-23 Revised:2025-09-23 Online:2025-12-01 Published:2025-11-27
Contact: Zhao Lei E-mail:2444713576@qq.com;zhaolei@scu.edu.cn
Supported by:
National Natural Science Foundation of China(81970944);Natural Science Foundation of Sichuan Province(2023NSFSC0553)

摘要/Abstract

摘要：

目的探究龈沟液（GCF）中细胞外游离DNA（cfDNA）与牙周临床指标、牙龈组织及牙龈成纤维细胞（HGFs）表达DNA感受通路环磷酸鸟苷—磷酸腺苷合成酶（cGAS）—干扰素基因刺激因子（STING）的可能关联。方法收集健康者和牙周炎患者的GCF及牙龈组织，记录受试者全口牙周临床指标，包括菌斑指数（PLI）、出血指数（BI）、探诊深度（PD）、临床附着水平（CAL），定量龈沟液cfDNA浓度，分析其与牙周临床指标的相关性；采用免疫荧光、定量逆转录聚合酶链反应（RT-qPCR）分别检测牙龈组织中cGAS、STING、p-STING的分布，以及cGAS-STING信号通路关键分子cGAS、STING、抑制因子激酶（IKK）、核因子κB p65（NF-κB p65）、白细胞介素（IL）-1β、IL-6、肿瘤坏死因子-α（TNF-α）mRNA的表达。应用健康组及牙周炎组GCF来源的cfDNA刺激HGFs，通过Western blot及RT-qPCR检测细胞cGAS-STING信号通路关键分子的表达。结果牙周炎组GCF中cfDNA水平显著高于健康组，且cfDNA水平与牙周临床指标呈强正相关性。与健康组相比，牙周炎组牙龈组织中cGAS、STING、p-STING与HGFs标志物FSP-1的荧光共定位百分比显著增加，牙周炎组牙龈组织中cGAS、STING、IKK、NF-κB p65、IL-1β、IL-6、TNF-α的mRNA表达水平高于对照组；与对照组相比，健康组或牙周炎组GCF来源cfDNA体外刺激HGFs后，cGAS和p-STING的蛋白表达水平呈升高趋势，cGAS、STING、IKK、NF-κB p65、IL-1β、IL-6、TNF-α的mRNA表达水平均显著增高。相关性分析显示：牙龈组织取样位点cfDNA浓度与cGAS、STING、NF-κB p65、IL-6的mRNA表达呈正相关性。结论牙周炎患者GCF中cfDNA水平显著升高，且可能通过激活HGFs的cGAS-STING信号通路在牙周炎进展中发挥重要作用。

关键词: 牙周炎, 细胞外游离DNA, 环磷酸鸟苷—磷酸腺苷合成酶—干扰素基因刺激因子信号通路, 人牙龈成纤维细胞

Abstract:

Objective This study aims to explore the potential relationships of cell-free DNA (cfDNA) in gingival crevicular fluid (GCF) with periodontal clinical indicators and the expression of DNA receptor pathway cyclic guanosine phosphate-adenosine phosphate synthase (cGAS)-stimulator of interferon genes (STING) in gingival tissues and human gingival fibroblasts (HGFs). Methods GCF and gingival tissue samples were collected from periodontally healthy individuals and patients diagnosed with periodontitis. Periodontal clinical indicators were recorded, including plaque index (PLT), bleeding index (BI), probing depth (PD), and clinical attachment level (CAL). The concentration of cfDNA in GCF was quantified, and the correlation between GCF and periodontal clinical indicators was analyzed. Immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to assess the distribution of cGAS, STING, and p-STING in gingival tissues. Additionally, the mRNA expression levels of the key components of the cGAS-STING signaling pathway, namely, cGAS, STING, inhibitory of kappa-B kinase (IKK), nuclear factor kappa-B p65 (NF-κB p65), interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), were measured. Furthermore, cfDNA extracted from GCF was employed to stimulate HGFs in the healthy control and periodontitis groups, and the mRNA expression levels of the key molecules of cGAS-STING signaling pathway were detected through Western blot and RT-qPCR. Results The concentration of cfDNA in GCF was found to be significantly elevated in the periodontitis group compared with the control group. Moreover, cfDNA concentration demonstrated a strong positive correlation with the periodontal clinical indicators. Immunofluorescence analysis revealed considerably increased percentage of fluorescence co-localization of cGAS, STING, and p-STING with the gingival fibroblast FSP-1 marker in the gingival tissues of the periodontitis group. The mRNA expression levels of cGAS, STING, IKK, NF-κB p65, IL-1β, IL-6,and TNF-α were significantly higher in the periodontitis group. In vitro stimulation of HGFs with GCF-derived cfDNA resulted in increased protein expression of cGAS and p-STING and considerably upregulated the mRNA expression levels of cGAS, STING, IKK, NF-κB p65, IL-1β, IL-6, and TNF-α in the healthy and periodontitis groups compared with the blank group. Correlation analysis showed that the concentration of cfDNA at the sampling site was positively correlated with the mRNA expression levels of cGAS, STING, NF-κB p65, and IL-6 in gingival tissues. Conclusion cfDNA concentrations in the GCF of patients with periodontitis are considerably elevated, and are associated with the activation of the cGAS-STING signaling pathway in HGFs. These findings suggest that cfDNA contributes to the progression of periodontitis.

Key words: periodontitis, cell-free DNA, cyclic guanosine phosphate-adenosine phosphate synthase-stimulator of interferon genes signaling pathway, human gingival fibroblasts

中图分类号:

R781.4

陈兰, 朱轩智, 周婕妤, 李继遥, 赵蕾. 龈沟液中细胞外游离DNA与牙周临床指标及环磷酸鸟苷—磷酸腺苷合成酶—干扰素基因刺激因子信号通路相关分子的关联性分析[J]. 华西口腔医学杂志, 2025, 43(6): 808-818.

Chen Lan, Zhu Xuanzhi, Zhou Jieyu, Li Jiyao, Zhao Lei. Correlation analysis of cell-free DNA in gingival crevicular fluid with periodontal clinical indicators and cyclic guanosine phosphate-adenosine phosphate synthase-stimulator of interferon genes signaling pathway[J]. West China Journal of Stomatology, 2025, 43(6): 808-818.

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参考文献 33

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基因	引物序列（5’-3’）	产物大小/bp
cGAS	F：GCTGCTCCACGAAGCCAAGAC	194
	R：GTCCACAACCCCTTTCACCATCC
STING	F：AGGAGAGCCACCAGAGCACAC	231
	R：ACCGCATTTGGGAGGGAGTAGTAG
IKK	F：GCGGTCCGACGTATGTGGTAAC	119
	R：TGTGCTGCTGCTGCGATGC
NF-κB p65	F：CGCATCCAGACCAACAACAACC	80
	R：TGGAAGCAGAGCCGCACAG
IL-6	F：GCCTTCGGTCCAGTTGCCTTC	133
	R：GTTCTGAAGAGGTGAGTGGCTGTC
TNF-α	F：TGGCGTGGAGCTGAGAGATAACC	149
	R：TCTGGTAGGAGACGGCGATGC
IL-1β	F：GGACAGGATATGGAGCAACAAGTGG	123
	R：CAACACGCAGGACAGGTACAGATTC
GAPDH	F：CACCCACTCCTCCACCTTTGAC	112
	R：GTCCACCACCCTGTTGCTGTAG

临床指标		健康组（n=10）	牙周炎组（n=10）
性别	男	5	4
	女	5	6
年龄/岁		24.00±1.48	30.20±3.87^***
BMI/（kg/m²）		21.06±1.72	21.53±2.19
PLI	全口	0.16±0.08	0.74±0.29^****
	取样位点	0.18±0.25	0.93±0.43^***
BI	全口	0.06±0.03	2.32±0.61^****
	取样位点	0.13±0.17	3.55±0.48^****
PD/mm	全口	1.90±0.19	4.40±0.85^****
	取样位点	2.35±0.25	9.35±0.98^****
CAL/mm	全口	0	4.31±1.57^****
	取样位点	0	8.95±1.81^****
GCF cfDNA/（mg/mL）		1.76±1.09	13.45±2.06^****

项目	年龄	BMI	PLI		BI		PD		CAL
项目	年龄	BMI	全口	取样位点	全口	取样位点	全口	取样位点	全口	取样位点
F值	15.750	0.258 0	16.140	12.440	80.000	97.990	88.810	133.100	115.100	173.000
r值	0.467	0.014 2	0.473	0.409	0.817	0.845	0.832	0.881	0.865	0.906
P值	0.000 9	0.617 4	0.000 8	0.002 4	<0.000 1	<0.000 1	<0.000 1	<0.000 1	<0.000 1	<0.000 1

组别	cGAS	STING	IKK	NF-κB p65	IL-1β	IL-6	TNF-α
健康组	1.06±0.40	0.77±0.24	1.69±1.48	0.88±0.15	1.21±0.71	1.15±0.77	0.79±0.33
牙周炎组	2.63±0.81^**	2.17±0.57^**	11.49±8.30^*	1.84±0.47^**	2.47±0.77^*	16.75±8.82^**	2.36±1.21^**

龈沟液中细胞外游离DNA与牙周临床指标及环磷酸鸟苷—磷酸腺苷合成酶—干扰素基因刺激因子信号通路相关分子的关联性分析

Correlation analysis of cell-free DNA in gingival crevicular fluid with periodontal clinical indicators and cyclic guanosine phosphate-adenosine phosphate synthase-stimulator of interferon genes signaling pathway

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