Bai X S, Lv L W, Zhou Y S
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.
Beijing Da Xue Xue Bao Yi Xue Ban. 2020 Feb 18;52(1):1-9. doi: 10.19723/j.issn.1671-167X.2020.01.001.
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.
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 polymerase 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.
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).
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.
明确 Tribbles 假激酶 3（TRIB3）在人脂肪间充质干细胞（hASCs）成脂分化过程中的作用，为 hASCs 在脂肪组织工程和软组织再生中的应用提供新靶点和新思路。
采用慢病毒转染技术构建 TRIB3 基因敲低的 hASCs（shTRIB3）和 TRIB3 过表达的 hASCs（TRIB3-over）。通过观察绿色荧光蛋白（GFP）在转染 hASCs 中的表达情况来评估转染效果。采用定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测慢病毒转染效率。成脂诱导后，通过油红 O 染色及定量分析，以及对几种特定成脂标志物进行 qRT-PCR，评估 hASCs 的成脂分化能力。
在 TRIB3 基因敲低的 hASCs 中，与对照组相比，TRIB3 mRNA 表达水平下降约 84.3%（P<0.01），TRIB3 蛋白水平也明显降低。相反，在 TRIB3 过表达的 hASCs 中，与对照组相比，TRIB3 mRNA 表达水平增加约 160%（P<0.01），TRIB3 蛋白水平也显著升高。这些结果表明成功构建了 TRIB3 基因敲低的 hASCs 和 TRIB3 过表达的 hASCs。油红 O 染色结果显示，TRIB3 下调显著促进 hASCs 中脂滴形成，与油红 O 定量分析结果一致。另一方面，TRIB3 上调抑制 hASCs 中脂滴形成，与油红 O 定量分析结果一致。成脂诱导后，与对照组相比，TRIB3 基因敲低的 hASCs 中过氧化物酶体增殖物激活受体γ（PPARγ）、分化抗原 36（CD36）和 CCAAT/增强子结合蛋白α（C/EBPα）等成脂相关基因显著增加（P<0.01）。相反，与对照组相比，TRIB3 过表达的 hASCs 中 PPARγ、CD36 和脂蛋白脂肪酶（LPL）显著降低（P<0.01）。
TRIB3 抑制 hASCs 的成脂分化。敲低 TRIB3 可促进 hASCs 的成脂能力，而过表达 TRIB3 则抑制 hASCs 的成脂分化。鉴于 PPARγ在成脂过程中的重要作用，TRIB3 调节功能的分子机制可能与 PPARγ信号通路有关。
