上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

人牙龈成纤维细胞复制性衰老模型的构建

夏一如，谢玉峰，束蓉   

  1. 上海交通大学 医学院附属第九人民医院牙周病科，上海市口腔医学重点实验室，上海 200011
  • 出版日期:2017-05-28 发布日期:2017-05-31
  • 通讯作者: 束蓉，电子信箱：shurong123@hotmail.com。
  • 作者简介:夏一如（1990—），女，硕士生；电子信箱：yiruxia@hotmail.com。
  • 基金资助:

    国家自然科学基金（81570977）；上海市教育委员会高峰高原学科建设计划

Establishment of a replicative senescence model of human gingival fibroblasts

XIA Yi-ru, XIE Yu-feng, SHU Rong   

  1. Shanghai Key Laboratory of Stomatology, Department of Periodontology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
  • Online:2017-05-28 Published:2017-05-31
  • Supported by:

    National Natural Science Foundation of China，81570977；Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support

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摘要：

目的 ·构建人牙龈成纤维细胞（hGFs）复制性衰老模型，探讨衰老相关生物学改变，为牙周疾病衰老相关研究提供有效的细胞模型。方法 ·取健康牙龈组织，利用组织块法体外培养hGFs，免疫荧光染色验证组织来源；连续传代培养，计算累积细胞群体倍增指数（CPD）并绘制曲线；取CPD值为10.82、20.65、29.52、42.22、60.79、70.03的hGFs，CCK-8试剂盒检测细胞增殖能力改变，real-time PCR检测衰老相关基因p16INK4a、p21Cip1的表达变化。结果 ·连续传代培养后，CPD值逐渐增大，达70.03时进入平台期；随着CPD值的增大，hGFs细胞形态呈扁平状，细胞突起增多；细胞增殖能力减弱；p16INK4a、p21Cip1 mRNA表达水平显著升高（P=0.000）。结论 ·连续传代培养可构建hGFs复制性衰老模型，CPD曲线可反映细胞衰老状态。

关键词: 牙龈, 细胞衰老, 组织块培养技术, 细胞增殖

Abstract:

Objective · To establish a replicative senescence model of human gingival fibroblasts (hGFs), investigate changes in aging related biological characteristics, and provide an efficient cell model for further study on the aging in periodontal diseases. Methods · hGFs were isolated from healthy gingival tissues and cultured with tissue block method in vitro. The tissue source was verified with immunofluorescence. hGFs were continuously cultured and cumulative population doublings (CPD) were calculated and used to draw the curves. Changes in the proliferative capacity of hGFs with CPDs of 10.82, 20.65, 29.52, 42.22, 60.79, and 70.03 were examined with CCK-8. Real-time PCR was used to evaluate changes in the mRNA expression of senescence-related genes p16INK4a and p21Cip1. Results · CPD curves showed that after continuous culture, the CPD value increased gradually and became stable after achieving 70.03. hGFs became flatter and more cell processes appeared with the increase of CPD value. The cell proliferative capacity declined and mRNA levels of p16INK4a and p21Cip1 significantly increased (P=0.000). Conclusion · A replicative senescence model of hGFs is established through continuous culture. CPD curves can reflect the aging of hGFs.

Key words: gingiva, cell aging, tissue block culture techniques, cell proliferation