CORM-3通过调控巨噬细胞极化促进MC3T3-E1细胞成骨分化的研究

doi:10.13701/j.cnki.kqyxyj.2022.03.017

口腔医学研究 ›› 2022, Vol. 38 ›› Issue (3): 280-285.DOI: 10.13701/j.cnki.kqyxyj.2022.03.017

CORM-3通过调控巨噬细胞极化促进MC3T3-E1细胞成骨分化的研究

王莹莹, 杜留熠, 吕慧欣, 王煜慧, 任思聪, 周延民^*

吉林大学口腔医院种植中心吉林长春 130021

收稿日期:2021-09-08 出版日期:2022-03-28 发布日期:2022-03-25
通讯作者: * 周延民,E-mail:zhouym@jlu.edu.cn
作者简介:王莹莹(1996~ ),女,甘肃人,硕士在读,医师,主要从事种植修复基础相关研究。
基金资助:
国家自然科学基金(编号:82071152)吉林省科技发展计划项目(编号:20180623061TC)

CORM-3 Promotes Osteogenic Differentiation of MC3T3-E1 Cells by Regulating the Polarization of Macrophages

WANG Yingying, DU Liuyi, LV Huixin, WANG Yuhui, REN Sicong, ZHOU Yanmin^*

Implant Center, Stomatology Hopspital, Jilin University, Changchun 130021, China

Received:2021-09-08 Online:2022-03-28 Published:2022-03-25

摘要/Abstract

摘要： 目的: 研究一氧化碳释放分子-3(carbon monoxide releasing molecule-3,CORM-3)对巨噬细胞极化的调控作用,进一步探究CORM-3介导的巨噬细胞免疫环境对MC3T3-E1细胞增殖和成骨分化的影响。方法: 体外培养RAW264.7细胞,筛选可用的CORM-3浓度。RT-qPCR检测iNOS、TGF-β、IL-10等M1/M2相关基因的表达。免疫荧光染色检测各组血红素加氧酶-1(heme oxygenase-1,HO-1)蛋白的表达。流式细胞术检测24 h后各组M1/M2巨噬细胞的比例。收集24 h后的上清液制备条件培养基,应用CCK-8、碱性磷酸酶染色和茜素红染色及RT-qPCR等检测MC3T3-E1细胞的增殖和成骨活性。结果: RT-qPCR结果显示CORM-3促进了TGF-β和IL-10的表达,降低了iNOS的表达(P<0.05),免疫荧光实验结果显示CORM-3增加RAW264.7细胞HO-1的蛋白表达,在CORM-3调控巨噬细胞的环境下,RT-qPCR结果显示CORM-3提高了OCN、Runx2及Col-1等基因的表达(P<0.05),MC3T3-E1的增殖和成骨分化增强。结论: CORM-3可通过释放CO提高HO-1的蛋白水平,促进巨噬细胞RAW264.7细胞向M2表型极化,增强MC3T3-E1 细胞的成骨分化能力。

关键词: 一氧化碳释放分子-3, 细胞极化, 条件培养基, 成骨分化

Abstract: Objective: To study the regulation of carbon monoxide releasing molecule-3 (CORM-3) on macrophage polarization, and to further explore the effects of CORM-3 mediated macrophage immune environment on proliferation and osteogenic differentiation of MC3T3-E1. Methods: RAW264.7 cells were cultured in vitro and the available CORM-3 concentration was screened. The expression of M1 and M2 related genes, such as iNOS, TGF-β, and IL-10, were detected by RT-qPCR. Immunofluorescence staining was used to detect the expression of Heme oxygenase-1 (HO-1) protein in each group. Flow cytometry was used to detect the proportion of M1 and M2 macrophages in each group after 24 hours. The supernatant after 24 hours was collected to prepare conditioned medium. CCK-8, alkaline phosphatase staining, alizarin red staining, and RT-qPCR were used to detect the proliferation and osteogenic activity of MC3T3-E1 cells. Results: RT-qPCR results showed that CORM-3 promoted the expression of TGF-β and IL-10, and decreased the expression of iNOS (P<0.05). Immunofluorescence results showed that CORM-3 increased the protein of HO-1 in RAW264.7 cells. In the environment where CORM-3 regulated macrophages, RT-qPCR results showed that CORM-3 increased the expression of OCN, Runx2, and Col-1 (P<0.05). The proliferation and osteogenic differentiation of MC3T3-E1 were enhanced. Conclusion: CORM-3 can increase the protein level of HO-1 by releasing CO, promote the polarization of macrophages RAW264.7 cells to the M2 phenotype, and enhance the osteogenic differentiation ability of MC3T3-E1 cells.

Key words: CORM-3, cell polarization, conditioned medium, osteogenic differentiation

王莹莹, 杜留熠, 吕慧欣, 王煜慧, 任思聪, 周延民. CORM-3通过调控巨噬细胞极化促进MC3T3-E1细胞成骨分化的研究[J]. 口腔医学研究, 2022, 38(3): 280-285.

WANG Yingying, DU Liuyi, LV Huixin, WANG Yuhui, REN Sicong, ZHOU Yanmin. CORM-3 Promotes Osteogenic Differentiation of MC3T3-E1 Cells by Regulating the Polarization of Macrophages[J]. Journal of Oral Science Research, 2022, 38(3): 280-285.

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CORM-3通过调控巨噬细胞极化促进MC3T3-E1细胞成骨分化的研究

CORM-3 Promotes Osteogenic Differentiation of MC3T3-E1 Cells by Regulating the Polarization of Macrophages

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