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浙江大学学报(医学版)  2021, Vol. 50 Issue (2): 171-178    DOI: 10.3724/zdxbyxb-2021-0109
专题报道     
牙龈卟啉单胞菌重组牙龈蛋白酶刺激牙龈成纤维细胞内钙离子浓度变化及其机制
张迪亚1(),卢可心2,李盛来3,吴燕岷2,*()
1.浙江大学医学院附属邵逸夫医院牙科,浙江 杭州 310016
2.浙江大学医学院附属第二医院牙周病专科,浙江 杭州 310009
3.浙江大学医学院附属口腔医院口腔外科,浙江 杭州 310006
Ca2+ mobilization and signaling pathways induced by Porphyromonas gingivalis rRgpB in human gingival fibroblast
ZHANG Diya1(),LU Kexin2,LI Shenglai3,WU Yanmin2,*()
1. Dental Department, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, China;
2. Department of Periodontology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China;
3. Oral Surgery, Stomatology Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China
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摘要:

目的:探讨牙龈卟啉单胞菌重组牙龈蛋白酶(rRgpB)刺激蛋白酶激活受体(PAR)介导的人牙龈成纤维细胞(HGF)内钙离子浓度([Ca2+]i)的动态变化及细胞内信号转导通路。方法:流式细胞术检测 HGF 表达的 PAR 类型,CCK-8 法检测细胞增殖,以牙龈卟啉单胞菌rRgpB作用于 HGF,激光共聚焦显微镜观察 HGF 内[Ca2+]i的动态变化及 PAR-1 拮抗剂的阻断作用;蛋白质印迹法检测 HGF 内 c-Jun 氨基末端激酶(JNK)、胞外信号调节激酶 1/2(ERK1/2)、p38 丝裂原激活的蛋白激酶(p38 MAPK)、p65 蛋白水平的变化。结果:HGF 表达 PAR-1 和 PAR-3,且 rRgpB 可促进 HGF 生长。细胞受到 rRgpB 作用后能激发瞬时增强的[Ca2+]i荧光信号,并且这种作用能够被 PAR-1 拮抗剂完全阻断;与空白对照组比较,rRgpB 诱导细胞6、12?h后,JNK、ERK1/2、p65 磷酸化蛋白表达均显著上调(均P<0.05),但 p38 MAPK 磷酸化蛋白水平未见明显变化(P>0.05);PAR-1 拮抗剂成功抑制了 rRgpB 诱导的 JNK、ERK1/2、p65 磷酸化蛋白表达的上调。结论:rRgpB 通过 PAR-1 诱导 HGF 内[Ca2+]i增加,并激活细胞内 JNK、ERK1/2、核因子κB 信号通路。

关键词: 牙龈蛋白酶人牙龈成纤维细胞蛋白酶激活受体钙离子信号通路    
Abstract:

Objective: To assess the Porphyromonas gingivalis (Pg) recombinant gingivalis gingipain R2 (rRgpB)-induced Ca2+ mobilization in human gingival fibroblast (HGF) mediated by protease-activated receptor (PAR) and its downstream signal transduction pathways. Methods: Flow cytometry was used to detect the expression of PAR in HGF. The proliferation of HGF was measured by CCK-8. The dynamic changes of intracellular Ca2+ concentration ([Ca2+]i) in HGF induced by rRgpB and the blocking effect of PAR-1 antagonist were observed by laser confocal microscopy. Western blot was performed to determine the phosphorylation levels of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38 MAPK) and p65 in HGF. Results: PAR-1 and PAR-3 were expressed in HGF, and the rRgpB could promote the proliferation of HGF. rRgpB caused a transient increase in [Ca2+]i, which could be completely suppressed by vorapaxar, a PAR-1 antagonist. The phosphorylation levels of JNK, ERK1/2 and p65 were significantly up-regulated after the induction of rRgpB for 6?h and 12?h (all P<0.05), which was completely inhibited by vorapaxar. However, the phosphorylation level of p38 MAPK had no significant change after rRgpB stimulation.Conclusions: rRgpB causes an increase in [Ca2+]i in HGF mediated by PAR-1. JNK, ERK1/2 and nuclear factor-κB may be involved in intracellular signal transduction after PAR-1 activation.

Key words: Gingipains    Human gingival fibroblasts    Protease-activated receptor    Calciumion    Signaling pathways
收稿日期: 2021-01-14 出版日期: 2021-06-18
CLC:  R781.4  
基金资助: 浙江省自然科学基金(LY17H140002); 浙江省新一轮卫生高层次人才培养工程
通讯作者: 吴燕岷     E-mail: zhangdiya@zju.edu.cn;wuyanmin@zju.edu.cn
作者简介: 张迪亚,副主任医师,主要从事牙周炎和种植体周围炎发病机制和治疗研究;E-mail:zhangdiya@zju.edu.cn;https://orcid.org/0000-0003-4471-9669. 卢可心,硕士研究生,主要从事牙周炎和种植体周围炎发病机制和治疗研究;E-mail:3130102947@zju.edu.cn;https://orcid.org/0000-0002-0924-9713
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引用本文:

张迪亚,卢可心,李盛来,吴燕岷. 牙龈卟啉单胞菌重组牙龈蛋白酶刺激牙龈成纤维细胞内钙离子浓度变化及其机制[J]. 浙江大学学报(医学版), 2021, 50(2): 171-178.

ZHANG Diya,LU Kexin,LI Shenglai,WU Yanmin. Ca2+ mobilization and signaling pathways induced by Porphyromonas gingivalis rRgpB in human gingival fibroblast. J Zhejiang Univ (Med Sci), 2021, 50(2): 171-178.

链接本文:

http://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0109        http://www.zjujournals.com/med/CN/Y2021/V50/I2/171

图 1  蛋白酶激活受体(PAR)在人牙龈成纤维细胞表面的表达结果
图 2  重组牙龈蛋白酶 R2(rRgpB)对人牙龈成纤维细胞增殖的影响与空白对照组比较,< 0.05,< 0.01.
图 3  各组人牙龈成纤维细胞内钙离子荧光图空白对照组细胞内钙离子荧光(绿色荧光)信号较弱,凝血酶组和 rRgpB 组细胞内钙离子荧光信号较强,rRgpB +沃拉帕沙组钙离子荧光信号强度与空白对照组相近. 标尺=20 μm.rRgpB:重组牙龈蛋白酶R2.
图 4  各组人牙龈成纤维细胞内钙离子荧光强度变化曲线rRgpB:重组牙龈蛋白酶R2.
图 5  重组牙龈蛋白酶 R2(rRgpB)刺激人牙龈成纤维细胞内信号通路相关蛋白表达的变化与空白对照组比较,< 0.05. JNK:c-Jun 氨基末端激酶;ERK:胞外信号调节激酶;GAPDH:甘油醛-3-磷酸脱氢酶.
图 6  沃拉帕沙对重组牙龈蛋白酶 R2(rRgpB)作用下人牙龈成纤维细胞内信号通路相关蛋白表达的影响与空白对照组比较,<0.05;与 rRgpB+沃拉帕沙组比较,< 0.05. JNK:c-Jun 氨基末端激酶;ERK:胞外信号调节激酶;GAPDH:甘油醛-3-磷酸脱氢酶.
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