巨噬细胞集落刺激因子过表达对信号通路中破骨相关因子的影响

doi:10.13701/j.cnki.kqyxyj.2019.11.019

口腔医学研究 ›› 2019, Vol. 35 ›› Issue (11): 1089-1093.DOI: 10.13701/j.cnki.kqyxyj.2019.11.019

巨噬细胞集落刺激因子过表达对信号通路中破骨相关因子的影响

王佳^1,2, 张文静^1,2, 韩祥祯^1,2, 周琦琪^1,2, 何惠宇^1,2*

1. 新疆医科大学第一附属医院(附属口腔医院)口腔修复科新疆乌鲁木齐 830054;
2. 新疆维吾尔自治区口腔医学研究所新疆乌鲁木齐 830054

收稿日期:2019-03-08 出版日期:2019-11-28 发布日期:2019-11-21
通讯作者: 何惠宇, E-mail: hehuiyu01@126.com
作者简介:王佳(1992~ ),女,新疆人,硕士在读,住院医师,从事口腔修复与种植方面相关临床及基础研究。
基金资助:
国家自然科学基金(编号:81660177)

Effect of Macrophage Colony-Stimulating Factor Overexpression on Osteoclast-related Cytokine Factors

WANG Jia^1,2, ZHANG Wenjing^1,2, HAN Xiangzhen^1,2, ZHOU Qiqi^1,2, HE Huiyu^1,2*

1. Department of Prosthodontics, the First Affiliated Hospital of Xinjiang Medical University (Affiliated Stomatological Hospital), Urumqi 830054, China;
2. Xinjiang Stomatological Research Institutes Urumqi 830054, China

Received:2019-03-08 Online:2019-11-28 Published:2019-11-21

摘要/Abstract

摘要： 目的: 探讨巨噬细胞集落刺激因子(monocyte colony-stimulating factor,M-CSF)在SD大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells ,BMSCs)中过表达对破骨细胞分化形成相关细胞因子在信号通路中表达的影响。方法: 构建过表达质粒载体上调M-CSF基因表达,使用增强型绿色荧光蛋白确定最佳转染剂量,并将质粒转染至BMSCs。采用逆转录-聚合酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR)检测破骨分化特异标志物 M-CSF、核因子-κB受体活化因子配体(receptor activator of nuclear factor-kappaB ligand,RANKL)、肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)、白细胞介素-1(interleukin-1,IL-1)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-24(interleukin-24,IL-24)的mRNA 表达水平。结果: 流式鉴定BMSCs 表面标记物CD29、CD44阳性率为91.4%、85.7%;CD45阳性率为2%;每孔5 μL LTX^®为最佳转染效率;RT-PCR检测结果显示,实验组BMSCs中M-CSF mRNA表达显著升高(P<0.001);BMSCs破骨分化特异标志物RANKL、TNF-α、IL-1 、IL-24 mRNA表达均显著增高(P<0.001),IL-6 mRNA表达降低(P<0.001),实验组与对照组比较差异有统计学意义。结论: M-CSF过表达增强BMSCs向破骨细胞分化的能力,对破骨细胞形成分化的早中晚期均存在影响。此外,M-CSF高表达质粒将外源基因导入BMSCs,是一种强有力的骨再生的基因治疗工具。

关键词: 巨噬细胞集落刺激因子, 破骨细胞相关因子, 破骨信号通路, 骨髓间充质干细胞, 骨组织工程

Abstract: Objective: To investigate the effect of macrophage colony-stimulating factor (M-CSF) overexpression in bone marrow mesenchymal stem cells (BMSCs) of SD rat on the osteoclast-associated cytokines. Methods: Overexpression plasmid vector to up-regulate M-CSF gene expression was constructed. The optimal transfection dose was determined using green fluorescent protein (EGFP) and the plasmid was transfected into BMSCs. The mRNA expression levels of osteoclast differentiation specific markers M-CSF, RANKL, TNF-α, IL-1, IL-6, and IL-24 were detected by reverse transcription polymerase chain reaction. Results: The positive rates of BMSCs surface markers CD29 and CD44 were 91.4% and 85.7% by flow cytometry and 2% by CD45. 5 μL/well LTX reagent was the best transfection efficiency. RT-PCR results showed that the expression of M-CSF in BMSCs of experimental group was significantly increased (P<0.001). The expression of RANKL, TNF-α, IL-1, and IL-24, the specific markers of osteoclast differentiation of BMSCs, increased significantly (P<0.001), while the expression of IL-6 decreased (P<0.001). There was a significant difference between the experimental group and the control group. Conclusion: Overexpression of M-CSF enhances the ability of BMSCs to differentiate into osteoclasts, and has an effect on the early, middle, and late stages of osteoclast differentiation. In addition, M-CSF high expression plasmid is a powerful gene therapy tool for bone regeneration by introducing foreign genes into BMSCs.

Key words: macrophage colony stimulating factor, osteoclast-related factors, osteoclastic signal pathway bone marrow mesenchymal stem cells, bone tissue engineering

王佳, 张文静, 韩祥祯, 周琦琪, 何惠宇. 巨噬细胞集落刺激因子过表达对信号通路中破骨相关因子的影响[J]. 口腔医学研究, 2019, 35(11): 1089-1093.

WANG Jia, ZHANG Wenjing, HAN Xiangzhen, ZHOU Qiqi, HE Huiyu. Effect of Macrophage Colony-Stimulating Factor Overexpression on Osteoclast-related Cytokine Factors[J]. Journal of Oral Science Research, 2019, 35(11): 1089-1093.

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巨噬细胞集落刺激因子过表达对信号通路中破骨相关因子的影响

Effect of Macrophage Colony-Stimulating Factor Overexpression on Osteoclast-related Cytokine Factors

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