地舒单抗对人脐带间充质干细胞增殖及成骨分化的作用研究

刘修志; 孟昊业; 赵金娟; 王鹏; 林万程; 王恺; 管延军; 张健; 许文静; 彭江

doi:10.3969/j.issn.2095-5227.2022.04.015

地舒单抗对人脐带间充质干细胞增殖及成骨分化的作用研究

Effects of denosumab on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells

摘要

摘要:
背景地舒单抗是一种RANKL抑制剂，具有抑制破骨细胞的作用。人脐带间充质干细胞(human umbilical cord mesenchymal stem cells，hUC-MSCs)具有成骨分化的能力。二者联合能否用于治疗股骨头坏死尚无定论。
目的研究地舒单抗对hUC-MSCs增殖及成骨分化的作用。
方法培养hUC-MSCs，使用生长状态良好的第3代hUC-MSCs进行实验。以含不同浓度地舒单抗(0.6 mg/mL、0.06 mg/mL、0.006 mg/mL)的hUC-MSCs成骨分化诱导培养基干预细胞作为实验组，以不含地舒单抗的hUC-MSCs成骨分化诱导培养基干预细胞作为对照组。使用CCK-8法检测不同组细胞的增殖活性；茜素红染色和碱性磷酸酶(alkaline phosphatase，ALP)染色分别检测各组细胞的成骨分化能力；实时荧光定量聚合酶链反应(qRT-PCR)检测各组细胞的ALP、锌指结构转录因子Osterix(OSX)、Ⅰ型胶原酶(type Ⅰ collagenase，COL-Ⅰ)、Runt相关转录因子2(Runt-related transcription factor 2，RUNX-2)的mRNA表达量。
结果通过流式细胞技术鉴定，hUC-MSCs可表达干细胞表面标志蛋白，且hUC-MSCs具有三系分化能力；与对照组相比，不同浓度地舒单抗(0.6 mg/mL、0.06 mg/mL、0.006 mg/mL)对hUC-MSCs具有促进增殖的作用，结果有统计学差异(P＜0.05)。当地舒单抗的浓度为0.006 mg/mL时，ALP/OSX/COL-Ⅰ/RUNX-2的表达量高于对照组(P＜0.05)；0.006 mg/mL地舒单抗处理的hUC-MSCs成骨分化能力较对照组增强(P＜0.05)，随着浓度的增加，hUC-MSCs的成骨分化能力降低，低于对照组，差异有统计学意义(P＜0.05)。
结论当地舒单抗的浓度为0.006 mg/mL时，能促进hUC-MSCs的增殖及成骨分化，高浓度(0.6 mg/mL、0.06 mg/mL)的地舒单抗抑制hUC-MSCs的成骨分化。

Abstract:
Background Denosumab is a RANKL inhibitor, which can inhibit osteoclasts. Human umbilical cord mesenchymal stem cells (hUC-MSCs) have the ability of osteogenic differentiation. Whether the combination of the two can be used in the treatment of femoral head necrosis is still uncertain.
Objective To investigate the effects of denosumab on the proliferation and osteogenic differentiation of hUC-MSCs.
Methods hUC-MSCs were cultured and the third generation of hUC-MSCs with good growth condition were used for experiment. The cultured cells treated with hUC-MSCs osteogenic differentiation induction medium containing various concentrations of denosumab (0.6 mg/mL, 0.06 mg/mL, 0.006 mg/mL) were served as experimental group and the cultured cells treated with hUC-MSCs osteogenic differentiation induction medium without denosumab as the control group. CCK-8 assay was used to detect the proliferation activity of different groups of cells. Alizarin red staining and alkaline phosphatase staining were used to detect the osteogenic differentiation ability of each group. The mRNA expression levels of alkaline phosphatase (ALP), zinc finger structural transcription factor (OSX), type Ⅰ collagenase (COL-Ⅰ) and Runt-associated transcription factor 2 (RUNX-2) were detected by real-time fluorescence quantitative polymerase chain reaction (QRT-PCR).
Results By flow cytometry identification, hUC-MSCs could express the surface marker proteins of stem cells, and hUC-MSCs had the ability of three-line differentiation. Compared with the control group, different concentrations of denosumab (0.6 mg/mL, 0.06 mg/mL, 0.006 mg/mL) promoted the proliferation of hUC-MSCs, and the results were statistically significant (P＜0.05). When the concentration of denosumab was 0.006 mg/mL, the expression levels of ALP/OSX/COL-I/ RUNX-2 were higher than those of the control group (P＜0.05). Osteogenic differentiation potential of hUC-MSCs treated by denosumab (0.006 mg/mL) was significantly higher than that of the control group (P＜0.05); With the increase of concentration, the osteogenic differentiation potential decreased, which was lower than that of the control group, and the differences were statistically significant (P＜0.05).
Conclusion Denosumab (0.006 mg/mL) can promote the proliferation and osteogenic differentiation of hUC-MSCs, while high concentrations of denosumab (0.6 mg/mL, 0.06 mg/mL) can inhibit the osteogenic differentiation of hUC-MSCs.

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