解剖学报 ›› 2022, Vol. 53 ›› Issue (1): 66-74.doi: 10.16098/j.issn.0529-1356.2022.01.009

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低氧化应激下肿瘤坏死因子α过表达对牙髓干细胞生物学性能的影响

李伟1 杨赣军2 彭建安2 胡红梅2* 周星辰1
  

  1. 1.井冈山大学医学部口腔系; 2.井冈山大学医学部组织学与解剖学教研室,江西 吉安 343000
  • 收稿日期:2020-11-08 修回日期:2021-01-06 出版日期:2022-02-06 发布日期:2022-02-06
  • 通讯作者: 胡红梅 E-mail:liwei8503@126.com
  • 基金资助:
    p38-MAPK和STAT3调控微氧化应激下TNF-α影响牙髓干细胞生物性能的分子机制研究;MCP-1/CCR2 轴介导 NF-κB 信号途径对牙髓损伤后牙髓干细胞迁移/归巢的影响

Effect of tumor necrosis factorα overexpress on the biological properties dental pulp stem cells in microoxidative stress

LI Wei YANG Gan-un PENG Jian-an2  HU Hong-mei2* ZHOU Xing-chen1    

  1. 1.Oral Department of Medical College, Jinggangshan University; 2.Department of Histology and Anatomy of  Medical college,Jinggangshan University, Jiangxi Ji’an 343000, China
  • Received:2020-11-08 Revised:2021-01-06 Online:2022-02-06 Published:2022-02-06
  • Contact: HU Hong-mei E-mail:liwei8503@126.com

摘要:

目的  探讨低氧化应激下肿瘤坏死因子α(TNF-α)过表达对牙髓干细胞(DPSCs)生物学性能的影响。   方法  实验分组为常氧DPSCs组、常氧空载转染组、常氧TNF-α过表达组、DPSCs 3% O2组、空载DPSCs 3% O2组、TNF-α过表达3% O2组。牙髓干细胞复苏并培养,然后将慢病毒TNF-α 转染细胞置于37 ℃、CO2培养箱培养, ELISA法检测各组细胞TNF-α的表达水平,RT-PCR检测TNF-α过表达氧化应激作用下的表达,Real-time PCR、Western blotting检测氧化应激作用下人DPSCs碱性磷酸酶(ALP)、牙本质涎磷蛋白(DSPP)表达和MTT检测各组细胞TNF-α转染后细胞增殖情况。   结果  Lentivirus-TNF-α转染细胞10 h 后,即可在倒置荧光显微镜下观察到绿色荧光信号,TNF-α过表达可以使低氧环境中牙髓干细胞增殖能力增强,且低氧的微环境可以增加TNF-α的表达,使DPSCs可以进一步的表达TNF-α,而且TNF-α过表达3% O2 时,Western blotting和Real-time PCR检测都表明DPSCs中ALP、DSPP表达差异具有显著性(P<0.05)。   结论  低氧微环境中DPSCs过表达TNF-α可以同时提高增殖效率和较高的分化效果。 

关键词: 低氧化, 肿瘤坏死因子-α, 牙髓干细胞, 免疫印迹法, 人 

Abstract:

Objective  To investigate the effect of tumor necrosis factor-α(TNF-α) on biological properties of dental pulp stem cells (DPSCs) in low-oxidation stress.    Methods  The experimental groups were as follows: normox, normox control plasmid, normox-TNF-α, 3% O2, control plasmid-3% O2、TNF-α-3% O2.DPSCs were resuscitated and cultured firstly, then lentivirus TNF-α transfected cells were cultured in a CO2 incubator at 37 ℃. The expression of TNF-α  was detected by ELISA in each group, and the expression of TNF-α under oxidative stress was detected by RT-PCR,the expressions of alkaline phosphatase(ALP) and dentin sialophosphoprotein(DSPP) were detected by Real-time PCR and Western blotting,the proliferation of DPSCs cells transfected with TNF-α was detected by flow cytometry and MTT.    Results  After lentivirus-TNF-α transfected cells for 10 hours, the green fluorescence signal could be observed under inverted fluorescence microscope, TNF-α overexpression could enhance the proliferation of DSPP in hypoxic environment,and hypoxia microenvironment can greatly increase the expression of TNF-α, Western blotting and Real-time PCR showed that there were significant differences in the expression of ALP and DSPP in DPSCs(P<0.05).    Conclusion  TNF-α overexpression can improve the proliferation efficiency and differentiation efficiency of DPSCs in hypoxic microenvironment. 

Key words: Micro oxidation, Tumor necrosis factor-α, Dental pulp stem cells, Western blotting, Human

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