上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2024 , Vol. 44 >Issue 2: 161 - 168

DOI: https://doi.org/10.3969/j.issn.1674-8115.2024.02.002

论著 · 基础研究

牙龈素提取物对口腔鳞癌细胞HN6生物学特性的影响

  • 李虎虓 ,
  • 李笑甜 ,
  • 赵旭日 ,
  • 张桓瑜 ,
  • 周薇 ,
  • 宋忠臣
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  • 1.上海交通大学医学院附属第九人民医院牙周病科，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011
    2.上海交通大学医学院附属第九人民医院口腔微生态与系统性疾病实验室，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海 200125
李虎虓（1994—），男，硕士，住院医师；电子信箱：tentigerli@163.com。
宋忠臣，电子信箱：szhongchen@sina.com。

收稿日期: 2023-09-20

  录用日期: 2023-12-26

  网络出版日期: 2024-02-28

基金资助

国家自然科学基金(82071112);上海交通大学医学院附属第九人民医院“交叉基金”(JYJC202005)

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Effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6

  • Huxiao LI ,
  • Xiaotian LI ,
  • Xuri ZHAO ,
  • Huanyu ZHANG ,
  • Wei ZHOU ,
  • Zhongchen SONG
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  • 1.Department of Periodontology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai 200011, China
    2.Laboratory of Oral Microbiota and Systemic Disease, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai 200125, China
SONG Zhongchen, E-mail: szhongchen@sina.com.

Received date: 2023-09-20

  Accepted date: 2023-12-26

  Online published: 2024-02-28

Supported by

National Natural Science Foundation of China(82071112);Cross-disciplinary Research Fund of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine(JYJC202005)

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摘要

目的·观察牙龈素提取物对口腔鳞状细胞癌（oral squamous cell carcinoma，OSCC）细胞HN6生物学特性的影响。方法·选取人OSCC细胞系HN6，加入牙龈卟啉单胞菌（Porphyromonas gingivalis，P. gingivalis）牙龈素提取物进行培养。根据牙龈素提取物的蛋白浓度不同分为对照组（control组），及牙龈素提取物蛋白浓度3.125 μg/mL组、6.25 μg/mL组、12.5 μg/mL组、25 μg/mL组、50 μg/mL组和100 μg/mL组，培养24、48 h后，采用细胞计数试剂盒8（cell counting kit-8，CCK-8）检测牙龈素提取物对HN6细胞增殖活性的影响。后续其他实验，分为control组、25 μg/mL组和50 μg/mL组进行。通过流式细胞术检测牙龈素提取物对细胞周期的影响，划痕实验和Transwell实验检测细胞迁移和侵袭能力，实时荧光定量PCR（real-time PCR，RT-PCR）和Western blotting检测细胞E-cadherin和N-cadherin蛋白和基因的表达。结果·在牙龈素提取物刺激HN6细胞24 h时，相比较control组，牙龈素提取物蛋白浓度25 μg/mL组（P=0.025），50 μg/mL组（P=0.000）和100 μg/mL组（P=0.049）的HN6细胞增殖活性显著增加；当刺激48 h时，6.25 μg/mL组（P=0.024）、12.5 μg/mL组（P=0.006）、25 μg/mL组（P=0.000）、50 μg/mL组（P=0.000）和100 μg/mL组（P=0.000）均较control组HN6细胞增殖活性有显著增加。细胞周期检测结果显示，与control组相比，经牙龈素提取物刺激24 h，HN6细胞G1期比例下降，S＋G2期比例显著性上升（25 μg/mL组：P=0.024；50 μg/mL组：P=0.001）。细胞迁移实验结果显示，与control组相比，随着牙龈素提取物浓度升高，划痕愈合的百分比显著增加（P=0.001）。细胞Transwell侵袭实验结果显示，与control组相比，随着牙龈素提取物浓度升高，细胞穿过小室底部的数量显著性增加。RT-PCR和Western blotting实验结果显示，与control组相比，随着牙龈素提取物浓度增加，HN6细胞中N-cadherin mRNA和蛋白表达量显著性增加，E-cadherin mRNA和蛋白表达量显著性减少。结论·牙龈素提取物对OSCC细胞HN6的增殖、迁移和侵袭有促进作用。

关键词： 牙龈素; 牙周炎; 口腔鳞状细胞癌; 细胞增殖; 细胞迁移; 细胞侵袭

本文引用格式

李虎虓 , 李笑甜 , 赵旭日 , 张桓瑜 , 周薇 , 宋忠臣 . 牙龈素提取物对口腔鳞癌细胞HN6生物学特性的影响[J]. 上海交通大学学报（医学版）, 2024 , 44(2) : 161 -168 . DOI: 10.3969/j.issn.1674-8115.2024.02.002

Abstract

Objective ·To observe the effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6. Methods ·The HN6 cell line was selected, cultivated, and divided into different groups based on the protein concentration of gingipain extract from Porphyromonas gingivalis: control group, 3.125 μg/mL group, 6.25 μg/mL group, 12.5 μg/mL group, 25 μg/mL group, 50 μg/mL group, and 100 μg/mL group. After 24 and 48 h of cultivation, CCK-8 assay was used to detect the effects of gingipain extract on HN6 cell proliferation activity. Subsequent experiments were divided into control group, 25 μg/mL group and 50 μg/mL group. Flow cytometry was used to examine the effects of gingipain extract on cell cycle. Scratch assay and Transwell assay were performed to evaluate cell migration and invasion ability. Real-time PCR (RT-PCR) and Western blotting were used to measure the expression of E-cadherin and N-cadherin proteins and genes in cells. Results ·Stimulated with gingipain extract for 24 h, the HN6 cells showed significantly increased proliferation activity in the 25 μg/mL (P=0.025), 50 μg/mL (P=0.000), and 100 μg/mL (P=0.049) groups compared to the control group. After 48 h, proliferation activity was significantly higher in the 6.25 μg/mL(P=0.024), 12.5 μg/mL (P=0.006), 25 μg/mL (P=0.000), 50 μg/mL (P=0.000), and 100 μg/mL (P=0.000) groups compared to the control group. Cell cycle analysis revealed that, after 24 h of gingipain stimulation, the proportion of HN6 cells in the G1 phase decreased, while the proportion in the S+G2 phase significantly increased compared to the control group (25 μg/mL group: P=0.024; 50 μg/mL group: P=0.001). Compared to the control group, the scratch assay demonstrated a significant increase in the percentage of scratch closure as the concentration of gingipain extract increased (P=0.001). Compared to the control group, the Transwell invasion assay showed a significant increase in the number of cells passing through the bottom of the chamber as the concentration of gingipain extract increased. RT-PCR and Western blotting results indicated that as the concentration of gingipain extract increased, the expression levels of N-cadherin mRNA and protein in HN6 cells significantly increased, while the expression levels of E-cadherin mRNA and protein significantly decreased compared to the control group. Conclusion ·Gingipain extract could promote proliferation, migration, and invasion of oral squamous cell carcinoma HN6 cells.

Key words： gingipain; periodontitis; oral squamous cell carcinoma (OSCC); cell proliferation; cell migration; cell invasion

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