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北京大学学报（医学版） ›› 2020, Vol. 52 ›› Issue (1): 1-9. doi: 10.19723/j.issn.1671-167X.2020.01.001

• 论著 •    下一篇

Tribbles同源蛋白3抑制人脂肪间充质干细胞成脂向分化

白向松,吕珑薇(),周永胜   

  1. 北京大学口腔医学院 ·口腔医院,修复科 国家口腔疾病临床医学研究中心 口腔数字化医疗技术和材料国家工程实验室 口腔数字医学北京市重点实验室,北京 100081
  • 收稿日期:2019-12-14 出版日期:2020-02-18 发布日期:2020-02-20
  • 通讯作者: 吕珑薇 E-mail:zhoucx2008@126.com
  • 基金资助:
    国家自然科学基金(31600787);中国科协青年人才托举项目(2015QNRC001);北京大学口腔医院青年基金(PKUSS20150107)

Tribbles pseudokinase 3 inhibits the adipogenic differentiation of human adipose-derived mesenchymal stem cells

Xiang-song BAI,Long-wei LV(),Yong-sheng ZHOU   

  1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
  • Received:2019-12-14 Online:2020-02-18 Published:2020-02-20
  • Contact: Long-wei LV E-mail:zhoucx2008@126.com
  • Supported by:
    Supported by the National Natural Science Foundation of China(31600787);the Young Elite Scientist Sponsorship Program by CAST(2015QNRC001);the Peking University School of Stomatology for Talented Young Investigators(PKUSS20150107)

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摘要：

目的:研究Tribbles同源蛋白3(Tribbles pseudokinase 3, TRIB3)在人脂肪间充质干细胞(human adipose-derived mesenchymal stem cells, hASCs)成脂分化中的调控作用,为提高hASCs成脂向分化能力、并为基于hASCs的脂肪组织工程与软组织缺损修复提供新靶点与新思路。方法:通过慢病毒转染建立TRIB3稳定敲低(TRIB3敲低组)和过表达(TRIB3过表达组)的hASCs细胞系,通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)和蛋白质印迹实验(Western blot)检测慢病毒转染hASCs的转染效率和效果。通过油红O染色和定量、qRT-PCR等实验方法,检测敲低、过表达TRIB3对hASCs成脂分化能力的影响。结果:TRIB3敲低慢病毒转染后,TRIB3敲低组hASCs中TRIB3 mRNA表达量较对照组下降84.3%(P<0.01), TRIB3蛋白表达也显著下降,表明成功构建了TRIB3敲低的hASCs细胞系;TRIB3过表达慢病毒转染后,TRIB3过表达组TRIB3 mRNA表达量较对照组增高约1.6倍(P<0.01),TRIB3蛋白表达也显著升高,表明成功构建了TRIB3过表达的hASCs细胞系。在此基础上,油红O染色显示,TRIB3敲低的hASCs成脂诱导后胞浆内红色脂滴明显增多,定量结果表明TRIB3敲低组油红O着色较对照组显著增多(P<0.01)。与之相反,TRIB3过表达的hASCs成脂诱导后胞浆内红色脂滴明显减少,定量结果表明TRIB3敲低组油红O着色较对照组显著减少(P<0.01)。qRT-PCR结果显示,敲低TRIB3的hASCs成脂诱导后,成脂分化相关基因过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor γ, PPARγ)、CCAAT增强子结合蛋白α(CCAAT/enhancer binding protein α, C/EBPα)和白细胞分化抗原36(cluster of differentiation 36, CD36)mRNA水平显著升高(P<0.01);TRIB3过表达的hASCs成脂诱导后成脂分化相关基因PPARγ、CD36和脂蛋白酯酶(lipoprotein lipase, LPL)mRNA水平明显下降。结论:TRIB3能够调控hASCs成脂分化能力,敲低TRIB3促进hASCs成脂分化,过表达TRIB3抑制hASCs成脂分化。

关键词: 人脂肪间充质干细胞, Tribbles同源蛋白3, 成脂分化

Abstract:

Objective: To identify the role of Tribbles pseudokinase 3 (TRIB3) during the process of adipogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs), and to provide a new target and a novel idea for the application of hASCs in adipose tissue engineering and soft tissue regeneration. Methods: TRIB3-knockdown hASCs (shTRIB3) and TRIB3-overexpression hASCs (TRIB3-over) were established using lentivirus transfection technique. The transfection effect was estimated by the visible presence of green fluorescence as the expression of green fluorescent protein (GFP) in the transfected hASCs. The lentiviral transfection efficiency was examined by quantitative real-time poly-merase chain reaction (qRT-PCR) and Western blot. After adipogenic induction, Oil Red staining and quantification, as well as qRT-PCR about several specific adipogenic markers were used to evaluate the adipogenic differentiation ability of hASCs. Results: In TRIB3-knockdown hASCs, the TRIB3 mRNA expression level decreased by about 84.3% compared with the control group (P<0.01), and the TRIB3 protein level also showed obvious reduction. Oppositely, in TRIB3-overexpression hASCs, the TRIB3 mRNA expression level increased by approximately 160% compared with the control group (P<0.01), and the TRIB3 protein level also showed a significant increase. These results indicated a successful construction of TRIB3-knockdown hASCs and TRIB3-overexpression hASCs. The Oil Red staining results showed that the down-regulation of TRIB3 significantly promoted lipid droplets formation in hASCs, consistent with Oil Red quantification. On the other hand, the up-regulation of TRIB3 suppressed lipid droplets formation in hASCs, consistent with Oil Red quantification. After adipogenic induction, adipogenesis-related genes, including peroxisome proliferator-activated receptor γ (PPARγ), cluster of differentiation 36 (CD36) and CCAAT/enhancer binding protein α (C/EBPα), were increased significantly in TRIB3-knockdown hASCs compared with the control group (P<0.01). Oppositely, PPARγ, CD36 and lipoprotein lipase (LPL) were significantly decreased in TRIB3-overexpression hASCs compared with the control group (P<0.01). Conclusion: TRIB3 inhibited the adipogenic differentiation of hASCs. Knockdown of TRIB3 promoted the ability of adipogenesis of hASCs, while overexpression of TRIB3 inhibited the adipogenic differentiation of hASCs. Considering the important role of PPARγ in the adipogenis process, the molecular mechanism of the regulatory function of TRIB3 may be related with PPARγ signal pathway.

Key words: Human adipose-derived mesenchymal stem cells, Tribbles pseudokinase 3, Adipogenic differentiation

中图分类号: 

  • R329.2

表1

慢病毒碱基序列"

表2

qRT-PCR引物序列"

Gene Forward primer (5' to 3') Reverse primer (5' to 3')
GAPDH CGGACCAATACGACCAAATCCG AGCCACATCGCTCAGACACC
TRIB3 GGGTCTGTTTTGCATGCGAGC AGCTCGTTTCTGGACGGGAC
PPARγ GAGGAGCCTAAGGTAAGGAG GTCATTTCGTTAAAGGCTGA
C/EBPα CGCAAGAGCCGAGATAAAGC CACGGCTCAGCTGTTCCA
LPL CGGATTAACATTGGAGAAGCTATCCG AGCTGGTCCACATCTCCAAGTC
CD36 CGATTAACATAAGTAAAGTTGCCATAATCG CGCAGTGACTTTCCCAATAGGAC

图1

shTRIB3和sh-NC转染hASCs后倒置荧光显微镜下观察(A)和TRIB3敲低效率qRT-PCR(B)、Western blot(C)检测"

图2

敲低TRIB3促进hASCs成脂分化的油红O染色(A)、油红O定量(B)和qRT-PCR(C)结果"

图3

TRIB3-over和NC-over转染hASCs后倒置荧光显微镜下观察(A)和TRIB3过表达效率qRT-PCR(B)和Western blot(C)检测"

图4

过表达TRIB3抑制hASCs成脂分化的油红O染色(A)、油红O定量(B)和qRT-PCR(C)结果"

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