MiR-210-3p转染对大鼠骨髓基质干细胞成骨活性影响△

Effect of Mir-210-3p transfection in rat bone marrow mesenchymal stem cells on their osteogenic capacity

摘要:

[目的] 探讨微小 RNA (microRNA, miR) -210-3p 转染大鼠骨髓基质干细胞 (bone marrow mesenchymal stem cells, BMMSCs) 对成骨活性的影响。[方法] 体外培养大鼠 BMMSCs，随机分 3 组，分别为空白对照组、miR-210-3p 增强组 (增强组)、miR-210-3p 抑制组(抑制组)，给予相应体外转染处理。检测细胞增殖、凋亡和迁移，以及诱导成骨分化和诱导成脂分化情况；采用 qRT-PCR 检测相应标志物的 mRNA 表达水平。[结果] 第 3 代 BMMSCs 的 CD44 和 CD90 呈阳性表达，CD34 和 CD45 呈阴性表达，符合间充质干细胞的表面抗原特征。与空白对照组对比，增强组的 miR-210-3p 的 mRNA 表达显著增加 (P＜0.05)，而抑制组的 miR-210-3p 的 mRNA 表达显著抑制(P＜0.05)。细胞增殖 OD 值、Transwell 细胞迁移、茜素红染色钙盐沉积计数由高至低依次均为增强组＞空白对照组＞抑制组(P＜0.05)；而凋亡率和油红 O 染色脂滴计数由低至高依次为增强组＜空白对照组＜抑制组(P＜0.05)。qRT-PCR 检测，成骨表达基因，包括 ALP 和 Bglap-2 的 mRNA 相对表达量由高至低依次为增强组＞空白对照组＞抑制组(P＜0.05)；而成脂表达基因，包括 PPARγ 和 LPL 的 mRNA 相对表达量由低至高依次为增强组＜空白对照组＜抑制组 (P＜0.05)。[结论] 本研究表明 miR-210-3p 可促进大鼠 BMMSCs 成骨分化，抑制成脂分化，减少细胞凋亡并促进细胞增殖和迁移。

Abstract:

[Objective] To explore the effect of microRNA (miR-210-3p) transfection in rat bone marrow mesenchymal stem cells (BMMSCs) on their osteogenic capacity. [Methods] Rat BMMSCs were isolated and cultured in vitro, and were randomly divided into 3 groups. The cells in the blank control group, MiR-210-3p enhanced group (enhanced group) and miR-210-3p inhibited group (inhibited group) were given transfection with the corresponding genes in vitro respectively. The cell biological behavious were detected, involving proliferation, apoptosis and migration, as well as osteogenic differentiation and adipogenic differentiation, in addition, qRT-PCR was used to detect mRNA expression levels of corresponding markers. [Results] The third generation BMMSCs obtained in this study were positive for CD44 and CD90, negative for CD34 and CD45, which were consistent with the surface antigen characteristics of mesenchymal stem cells. Compared with blank control group, mRNA expression of miR-210-3p in enhanced group was significantly increased (P＜0.05), whereas which in the inhibition group was significantly inhibited (P＜0.05). The OD value of cell proliferation, Transwell cell migration and alizarin red staining calcium deposition count were significantly ranked from high to low as the enhanced group ＞ blank control group＞inhibition group (P＜0.05). Conversely, the apoptosis rate and oil red O staining lipid drop count were significantly ranked from low to high as the enhanced group＜blank control group＜ inhibition group (P＜0.05), As results of qRT-PCR assay, the mRNA relative expression level of osteo-genic genes, including ALP and Bglap-2, was significantly ranked from high to low as the enhanced group＞blank control group＞inhibitiongroup (P＜0.05) , whereas the mRNA relative expression levels of lipid-expressing genes, including PPARγ and LPL, were from low to highas the enhanced group＜blank control group＜inhibition group (P＜0.05) . [Conclusion] In this study the miR-210-3p does promote osteogen-ic differentiation, inhibit adipogenic differentiation, reduce apoptosis, and promote cell proliferation and migration of BMMSCs in rats.