摘要：

目的 ：探究microRNA-143-3p（miR-143-3p）对人牙髓干细胞（human dental pulp stem cells，hDPSCs）凋亡及分化能力的影响及其作用机制。方法： 分离hDPSCs，并转染miR-143-3p抑制物（inhibitor）或核因子κB受体活化因子（receptor activator of nuclear factor-κB，RANK）小干扰RNA（small interfering RNA，siRNA），通过流式细胞术检测细胞凋亡情况，对细胞进行成骨分化诱导后通过茜素红染色评估分化情况，分别用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR) 和蛋白质印迹法(Western blotting)检测细胞骨保护素（osteoprotegerin，OPG）、RANK、核因子κB受体活化因子配体（receptor activator of nuclear factor-κB ligand，RANKL）、Runt相关转录因子2（Runt-related transcription factor 2，Runx2）、骨钙素（osteocalcin，OCN）、碱性磷酸酶（alkaline phosphatase，ALP）、骨形态发生蛋白2（bone morphogenetic protein 2，BMP2）mRNA及蛋白表达，双荧光素酶实验分析miR-143-3p与RANK的靶向关系。结果： miR-143-3p在hDPSCs中的表达与RANK呈反向调控关系，抑制miR-143-3p的表达增加了细胞凋亡率，促进了细胞成骨分化，提高了RANK、RANKL、Runx2、OCN、ALP、BMP2 的mRNA和蛋白表达水平，降低了OPG的mRNA和蛋白表达水平（P<0.05），而沉默RANK则抑制了miR-143-3p inhibitor的作用（P<0.05）。双荧光素酶实验提示，RANK是miR-143-3p的靶基因。结论： miR-143-3p通过靶向RANK激活OPG/RANKL轴调节hDPSCs的凋亡及成骨分化。

关键词: 人牙髓干细胞, miR-143-3p, 核因子κB受体活化因子, 凋亡, 分化

Abstract:

Objective: To explore the effect and mechanism of miR-143-3p on the apoptosis and differentiation of human dental pulp stem cells(hDPSCs). Methods: hDPSCs were isolated, and transfected with miR-143-3p inhibitor, or receptor activator of nuclear factor-κB (RANK), small interfering RNA (siRNA). Cell apoptosis was detected by flow cytometry. After inducing hDPSCs into osteogenic differentiation, the cellular differentiation was evaluated by alizarin red staining. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect cellular mRNA and protein expression levels of osteoprotegerin (OPG), RANK, and receptor activator of nuclear factor-κB ligand (RANKL), Runt-related transcription factor 2(Runx2), osteocalcin(OCN), alkaline phosphatase(ALP), and bone morphogenetic protein 2 (BMP2). Dual luciferase reporter assay was used to analyze the targeting relationship between miR-143-3p and RANK. Results: The expression of miR-143-3p in hDPSCs was inversely regulated by RANK. Inhibiting the expression of miR-143-3p significantly increased the rate of cell apoptosis, promoted the osteogenic differentiation of cells, increased the mRNA and protein expression levels of RANK, RANKL, Runx2, OCN, ALP, BMP2, and decreased the mRNA and protein expression levels of OPG (all P<0.05), but silence of RANK inhibited the effect of miR-143-3p inhibitor (P<0.05). Dual luciferase reporter assay indicated that RANK was the target gene of miR-143-3p. Conclusion: MiR-143-3p activitates the OPG/RANKL axis and thus regulates the apoptosis and osteogenic differentiation of hDPSCs by targeting RANK.

Key words: human dental pulp stem cells, miR-143-3p, receptor activator of nuclear factor-κB, apoptosis, differentiation

中图分类号: 

• R782