Rorβ 在激活的巨噬细胞和牙髓干细胞中的表达。

Ror β expression in activated macrophages and dental pulp stem cells.

机构信息

College of Dental Medicine, University of Sharjah, Sharjah, UAE.

Sharjah Institute for Medical Research, University of Sharjah, Sharjah, UAE.

出版信息

Int Endod J. 2021 Mar;54(3):388-398. doi: 10.1111/iej.13431. Epub 2020 Nov 18.

DOI:10.1111/iej.13431

PMID:33075145

Abstract

AIM

To assess the expression of Retinoic acid-related orphan receptor beta (Ror β) in human inflamed dental pulp stem cells (hI-DPSCs) and during macrophage phenotypic conversion.

METHODOLOGY

Commercially procured THP-1 monocytes conversion to macrophages was judged by their morphology, the percentage of adherent cells and the expression of CD-14 surface marker. THP-1 macrophage cell viability following LPS, IFN-γ/IL-4, IL-13 stimulus was evaluated at 24 and 48h. The phenotypic conversion of macrophages to M1 and M2 was confirmed by flow cytometry and Western blot analysis. Cytokine release following polarization was estimated by the BD cytokine flex kit. The expression of Ror β in THP-1 macrophages and hI-DPSCs following LPS, IFN-γ/IL-4, IL-13 stimulus was assessed by Western blot analysis. Statistical significance was analysed using one-way Anova followed by Tukey's Post hoc test.

RESULTS

THP-1 monocytes pretreated with PMA (100 ng mL ) for 48 h followed by culturing in PMA-free media for another 48 h yielded cells with morphological characteristics similar to macrophages with a high percentage of adherence capability and CD-14 expression. Macrophages treated with LPS 100 ng mL and IFN-γ 20 ng mL or IL-4 20 ng mL had high expression of the respective M1 and M2 CD markers in flow cytometry and Western blot analysis. Cytokine release studies demonstrated the expression of IL-1β, TNF-α and IL-10 in the M1-polarized macrophages (P < 0.01), whilst TGF- β levels were seen in the M1 and M2-polarized macrophages. Ror β expression was upregulated when macrophages and hI-DPSCs were treated with anti-inflammatory cytokines.

CONCLUSION

Ror β was expressed in THP-1 macrophages and hI-DPSCs during their resting stage. Upregulated expression of Ror β occurred following an anti-inflammatory stimulus.

摘要

目的

评估维甲酸相关孤儿受体β（Ror β）在人牙髓干细胞（hI-DPSCs）炎症和巨噬细胞表型转化过程中的表达。

方法

通过形态学、贴壁细胞百分比和 CD-14 表面标志物的表达判断商业采购的 THP-1 单核细胞向巨噬细胞的转化。用 LPS、IFN-γ/IL-4、IL-13 刺激 24 和 48 小时后，评估 THP-1 巨噬细胞的活力。通过流式细胞术和 Western blot 分析确认巨噬细胞向 M1 和 M2 的表型转化。用 BD 细胞因子 flex 试剂盒估计极化后细胞因子的释放。用 Western blot 分析 LPS、IFN-γ/IL-4、IL-13 刺激后 THP-1 巨噬细胞和 hI-DPSCs 中 Ror β 的表达。采用单因素方差分析（one-way Anova），然后进行 Tukey 事后检验，分析统计学意义。

结果

用 PMA（100ng/ml）预处理 THP-1 单核细胞 48 小时，然后在无 PMA 培养基中培养 48 小时，得到具有类似巨噬细胞形态特征的细胞，贴壁能力高，CD-14 表达高。用 LPS 100ng/ml 和 IFN-γ 20ng/ml 或 IL-4 20ng/ml 处理的巨噬细胞，在流式细胞术和 Western blot 分析中，M1 和 M2 CD 标志物的表达较高。细胞因子释放研究表明，M1 极化巨噬细胞中表达了 IL-1β、TNF-α 和 IL-10（P<0.01），而 TGF-β 水平见于 M1 和 M2 极化巨噬细胞中。用抗炎细胞因子处理巨噬细胞和 hI-DPSCs 时，Ror β 的表达上调。