国际口腔医学杂志

国际口腔医学杂志 ›› 2020, Vol. 47 ›› Issue (3): 286-292.doi: 10.7518/gjkq.2020062

• 论著 • 上一篇    下一篇

人单核细胞和外周血单个核细胞衍生的巨噬细胞极化特性的比较

刘晔1,2,洪润丹2,王志国3,刘涵云4,孟琛达2,王茹2,徐全臣1()   

  1. 1.青岛大学附属医院口腔内科 青岛 266003;
    2.青岛大学口腔医学院 青岛 266003;
    3.青岛大学附属医院美容整形科 青岛 266003;
    4.青岛大学附属医院感染性疾病科 青岛 266003
  • 收稿日期:2019-09-17 修回日期:2019-11-22 出版日期:2020-05-01 发布日期:2020-05-08
  • 通讯作者: 徐全臣
  • 作者简介:刘晔,硕士,Email：lyly_1206@163.com
  • 基金资助:
    国家自然科学基金(81500849);山东省重点研发计划(2018GSF118150);山东省自然科学基金(ZR2017MH083)

Comparison of polarization characteristics of human monocyte cell- and peripheral blood mononuclear cell-derived macrophages

Liu Ye1,2,Hong Rundan2,Wang Zhiguo3,Liu Hanyun4,Meng Chenda2,Wang Ru2,Xu Quanchen1()   

  1. 1.Dept. of Oral Medicine, The Affiliated Hospital of Qingdao University, Qingdao 266003, China;
    2.School of Stomatology, Qingdao University, Qingdao 266003, China;
    3.Dept. of Burn and Plastic Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266003, China;
    4.Dept. of Infectious Diseases, The Affiliated Hospital of Qingdao University, Qingdao 266003, China
  • Received:2019-09-17 Revised:2019-11-22 Online:2020-05-01 Published:2020-05-08
  • Contact: Quanchen Xu
  • Supported by:
    National Natural Science Foundation of China(81500849);Shandong Province Key Research Plan(2018GSF118150);Shandong Provincial Natural Science Foundation(ZR2017MH083)

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摘要：

目的 在成功建立巨噬细胞（Mφ）体外培养模型基础上,比较不同来源Mφ的极化特性。方法 采用密度梯度离心法和免疫磁珠法体外分离并培养外周血单个核细胞（PBMC）,经人重组巨噬细胞集落刺激因子诱导后培养出PBMC Mφ。人单核细胞系THP-1在100 nmol·L-1佛波酯刺激下分化为THP-1 Mφ。上述两种来源的Mφ分别采用不同的极化因子进行诱导,经50 ng·mL-1脂多糖+20 ng·mL-1干扰素-γ诱导形成M1型Mφ（M1 Mφ）,经20 ng·mL-1白细胞介素-4诱导形成M2型Mφ（M2 Mφ）。观察两种来源Mφ的细胞形态,比较两种细胞表面极化标记物（CD86和CD206）和细胞因子[白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、甘露糖受体C1样蛋白（MRC1）、白细胞介素-10（IL-10）等]的表达情况。结果 两种来源的Mφ经M1极化因子刺激后,TNF-α、IL-1β的mRNA水平显著上调,CD86表达量增加,与PBMC Mφ相比,THP-1 Mφ中的TNF-α、IL-1β分泌水平更高;两种Mφ在M2极化因子刺激后MRC1、IL-10 mRNA水平上调,CD206在PBMC Mφ中表达显著增强,而在THP-1 Mφ中表达没有明显变化。结论 THP-1可能更适合进行M1极化的研究,而PBMC可能更适合进行M2极化的研究。

关键词: 巨噬细胞极化, 人单核细胞, 外周血单个核细胞

Abstract:

Objective Obtained from successfully established polarization models, the polarization characteristics of macrophages (Mφ) from different sources were compared to provide an experimental basis for biological treatments. Methods Peripheral blood mononuclear cells (PBMC) were isolated through density gradient centrifugation and magnetic cell sorting and were cultured in vitro. PBMC Mφ was induced by human recombinant macrophage colony-stimulating factor. The human monocyte cell line THP-1 was differentiated into THP-1 Mφ under the induction of 100 nmol·L-1 phorbol 12-myristate 13-acetate. Morphology, polarization marker expression (CD86 and CD206), and cytokine secretion levels of the two groups were compared under the condition of M1 or M2 polarization factors. M1 polarization factors was 50 ng·mL-1 lipopolysaccharides and 20 ng·mL-1 interferon-γ, and M2 polarization factors was 20 ng·mL-1 interleukin-4. Results After M1 polarization, the mRNA levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were markedly upregulated, the expression of CD86 was enhanced, and the secretions of IL-1β and TNF-α in THP-1 Mφ were higher than those in PBMC Mφ. After M2 polarization, the mRNA levels of mannose receptor C-type 1 and interleukin-10 increased, and the expression of CD206 was upregulated in PBMC Mφ. However, no difference was observed in THP Mφ. Conclusion THP-1 is appropriate for M1 polarization, and PBMC is appropriate for M2 polarization.

Key words: macrophage polarization, human monocyte cell, peripheral blood mononuclear cell

中图分类号: 

  • Q25

图 1

密度梯度离心法中液体分为3层 上层：血浆和PBS液;中层：淋巴细胞分离液;下层：红细胞和粒细胞;单个核细胞层：上、中层界面处的白色云雾层狭窄带。"

表 1

qRT-PCR检测基因的引物序列"

基因名称 引物序列（5’—3’）
IL-1β 正向：TGGCTTATTACAGTGGCAATGAGGATG
反向：TGTAGTGGTGGTCGGAGATTCGTAG
TNF-α 正向：AGCTGGTGGTGCCATCAGAGG
反向：TGGTAGGAGACGGCGATGCG
MRC1 正向：TTGGACGGATGGACGAGGAGTC
反向：CGGATCGTGTCTGGCATATGTAGC
IL-10 正向：GCCAAGCCTTGTCTGAGATGATCC
反向：GCTCCACGGCCTTGCTCTTG
GAPDH 正向：TGCACCACCAACTGCTTAGC
反向：GGCATGGACTGTGGTCATGAG

图 2

两种来源Mφ的形态学变化 倒置显微镜 A：静息状态下的PBMC;B：静息状态下的THP-1;C：rhM-CSF诱导下分化培养7 d后的PBMC Mφ;D：PMA诱导24 h后的THP-1 Mφ;E：极化因子刺激后的PBMC M1;F：极化因子刺激后的THP-1 M1。"

图 3

两种来源Mφ极化后细胞因子mRNA的相对表达量 A：IL-1β;B：TNF-α;C：MRC1;D：IL-10。?,P<0.05。"

图 4

Mφ极化标记物CD86和CD206的表达 上：CD86表达;下：CD206表达;M0：静息状态;M1：M1极化状态;M2：M2极化状态。?,P<0.05。"

表 2

两种来源Mφ上清液中细胞因子的表达量"

细胞因子 对照组（静息状态） LPS+IFN-γ刺激 IL-4刺激
THP-1 PBMC THP-1 PBMC THP-1 PBMC
IL-1β 58.43±1.83 56.25±1.74 591.35±5.67 551.73±8.51 62.58±1.97 60.72±1.81
TNF-α 41.57±2.35 39.16±2.27 436.91±10.72 338±13.51 45.37±2.48 41.42±2.71
IL-6 28.73±1.98 47.39±2.31 307.59±9.53 429.86±11.62 36.26±1.76 49.98±2.74
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