沉默Foxp3对牙周膜成纤维细胞在炎症环境下炎症因子的表达及增殖和迁移的影响

doi:10.7518/hxkq.2023.2022409

华西口腔医学杂志 ›› 2023, Vol. 41 ›› Issue (3): 269-275.doi: 10.7518/hxkq.2023.2022409

沉默Foxp3对牙周膜成纤维细胞在炎症环境下炎症因子的表达及增殖和迁移的影响

芦婷¹(), 朱嘉皓¹, 杨仕鹤¹, 沈喆¹, 钟良军¹^,²()

^1.杭州师范大学口腔医学院，杭州 310000
^2.杭州师范大学附属医院牙周病诊疗中心，杭州 310000

收稿日期:2022-10-18 修回日期:2022-12-31 出版日期:2023-06-01 发布日期:2023-06-02
通讯作者: 钟良军 E-mail:1813253860@qq.com;zymdxx@163.com
作者简介:芦婷，住院医师，硕士，E-mail：1813253860@qq.com
基金资助:
国家自然科学基金(81570989);杭州市卫生科技计划重大项目(Z20200046);杭州市医药卫生科技重点项目(ZD-20210016);浙江省教育厅一般项目(Y202147690)

Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment

Lu Ting¹(), Zhu Jiahao¹, Yang Shihe¹, Shen Zhe¹, Zhong Liangjun¹^,²()

^1.School of Stomatology, Hangzhou Normal University, Hangzhou 310000, China
^2.Dept. of Periodontal Clinic, The Affiliated Hospital of Hangzhou Normal University, Hangzhou 310000, China

Received:2022-10-18 Revised:2022-12-31 Online:2023-06-01 Published:2023-06-02
Contact: Zhong Liangjun E-mail:1813253860@qq.com;zymdxx@163.com
Supported by:
The National Natural Science Foundation of China(81570989);Hangzhou Municipal Health Science and Technology Plan Major Project(Z20200046);Hangzhou Medical and Health Science and Technology Key Projects(ZD20210016);General Project of Department of Education of Zhejiang Provincial(Y202147690)

摘要/Abstract

摘要：

目的探究炎症环境下Foxp3基因对人牙周膜成纤维细胞（hPDLFs）炎症因子表达及细胞增殖、迁移的影响，探索Foxp3基因在牙周炎发生发展中的作用。方法使用小干扰RNA（siRNA）沉默hPDLFs的Foxp3基因，通过逆转录聚合酶链反应（RT-PCR）、免疫印迹（Western blotting）实验验证Foxp3沉默效率，筛选Foxp3基因沉默效果最佳的siRNA。使用牙龈卟啉单胞菌（P. gingivalis）来源脂多糖（LPS）模拟体外炎症环境，CCK-8检测炎症环境下沉默Foxp3对hPDLFs增殖的影响；划痕实验及transwell细胞迁移实验检测炎症环境下沉默Foxp3对hPDLFs迁移的影响；RT-PCR、Western blotting实验检测炎症环境下hPDLFs炎症因子白细胞介素（IL）-6、IL-8的表达。结果 siRNA转染后，RT-PCR、Western blotting检测显示，Foxp3-si3组Foxp3的mRNA和蛋白表达均显著降低（mRNA表达，t=21.03，P<0.000 1；蛋白表达，t=12.8，P<0.001）。在炎症环境下，Foxp3基因沉默对hPDLFs的增殖无显著影响（P>0.05）；Foxp3基因沉默促进hPDLFs的迁移，IL-6、IL-8的表达升高（P<0.05）。结论在炎症环境下，Foxp3基因沉默可促进hPDLFs迁移和炎症因子表达升高，但对hPDLFs的增殖无明显影响。Foxp3基因在牙周炎中具有抑制炎症的作用。

关键词: 牙周膜成纤维细胞, Foxp3, 脂多糖, 小干扰RNA, 牙周炎

Abstract:

Objective This study aimed to clarify the effects of Foxp3 silencing on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLFs) in an inflammatory environment and on cell proliferation and invasiveness, as well as to explore the role of Foxp3 gene in the development of periodontitis. Methods An small interfering RNA (siRNA) construct specific for Foxp3 was transfected into hPDLFs. Foxp3 silencing efficiency was verified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and the siRNA with the optimum silencing effect of Foxp3 gene was screened. Using lipopolysaccharide to simulate an inflammatory environment in vitro, CCK-8 detected the effect of silencing Foxp3 on hPDLFs proliferation under inflammatory conditions. Wound-healing experiments and transwell assays were conducted to detect the effect of silencing Foxp3 on hPDLF migration under inflammatory conditions. The expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 was detected by RT-PCR and Western blotting under inflammatory conditions. Results After siRNA transfection, RT-PCR and Western blotting analyses showed that the expression of Foxp3 mRNA in the Foxp3-si3 group decreased significantly (t=21.03, P<0.000 1), and the protein expression of Foxp3 also decreased significantly (t=12.8, P<0.001). In the inflammatory environment, Foxp3 gene silencing had no significant effect on hPDLFs proliferation (P>0.05), and Foxp3 gene silencing promoted hPDLFs migration (P<0.05). Moreover, the expression of IL-6 and IL-8 increased (P<0.05). Conclusion In an inflammatory environment, Foxp3 gene silencing promoted hPDLFs migration but had no significant effect on hPDLFs proliferation. The expression of inflammatory factors expressed in hPDLFs increased after Foxp3 gene silencing, indicating that Foxp3 gene inhibited inflammation in periodontitis.

Key words: human periodontal ligament fibroblasts, Foxp3, lipopolysaccharide, small interfering RNA, perio-dontitis

中图分类号:

R 783.5

芦婷, 朱嘉皓, 杨仕鹤, 沈喆, 钟良军. 沉默Foxp3对牙周膜成纤维细胞在炎症环境下炎症因子的表达及增殖和迁移的影响[J]. 华西口腔医学杂志, 2023, 41(3): 269-275.

Lu Ting, Zhu Jiahao, Yang Shihe, Shen Zhe, Zhong Liangjun. Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment[J]. West China Journal of Stomatology, 2023, 41(3): 269-275.

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引物	序列（5’-3’）
NC	F：UUCUCCGAACGUGUCACGUdTdT R：ACGUGACACGUUCGGAGAAdTdT
Foxp3-si1	F：GGAUGUGAGAAGGUCUUCGAA R：UUCGAAGACCUUCUCACAUCC
Foxp3-si2	F：GCACAUUCCCAGAGUUCCU R：AGGAACUCUGGGAAUGUGC
Foxp3-si3	F：CACUCAAUGAGAUCUACCA R：UGGUAGAUCUCAUUGAGUG

引物	序列（5’-3’）
18s	F：TAGAGGGACAAGTGGCGTTC R：CGCTGAGCCAGTCAGTGT
IL-6	F：CTGGATTCAATGAGGAGACTTGCC R：GGTTGGGTCAGGGGTGGTTA
IL-8	F：ACTCCAAACCTTTCCACC R：CTTCTCCACAACCCTCTG
Foxp3	F：CATCTGTGGCATCATCCGAC R：GTGGAGGAACTCTGGGAATGT

组别	样本量	Foxp3
NC	3	1.001±0.051 42
Foxp3-si1	3	0.592 6±0.081 35
Foxp3-si2	3	0.880 3±0.026 68
Foxp3-si3	3	0.184 4±0.043 35^#

组别	样本量	IL-6	IL-8
NC-LPS	3	1.029±0.280 5	1.013±0.193 8
Foxp3-si3-LPS	3	2.166±0.530 8	1.657±0.314 1
t值		3.282	3.020
P值		0.030 4	0.039 2

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沉默Foxp3对牙周膜成纤维细胞在炎症环境下炎症因子的表达及增殖和迁移的影响

Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment

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