含硅羟基磷灰石调节巨噬细胞极性促进成骨分化机制研究  

Study on the mechanism of silicon-containing hydroxyapatite regulating macrophage polarity and promoting osteogenesis differentiation

作　　者：王志英[1] 杨秋霞[2] 田新利[3] 林雪霞[4] WANG Zhi-ying;YANG Qiu-xia;TIAN Xin-li;LIN Xue-xia(Department of Pathology,Xingtai Medical College,Xingtai 054000,China;Department of Prevention Medicine,Xingtai Medical College,Xingtai 054000,China;Department of Immunology,Xingtai Medical College,Xingtai 054000,China;Department of Physiology,Xingtai Medical College,Xingtai 054000,China)

机构地区：[1]河北邢台医学高等专科学校病理教研室,054000 [2]河北邢台医学高等专科学校预防医学教研室,054000 [3]河北邢台医学高等专科学校微生物免疫教研室,054000 [4]河北邢台医学高等专科学校生理教研室,054000

出　　处：《天津医药》2021年第9期910-916,共7页Tianjin Medical Journal

基　　金：邢台市科技计划项目(2018ZC101)。

摘　　要：目的探讨无机骨再生修复材料含硅羟基磷灰石(si-HA)通过调节巨噬细胞极性转换促进小鼠前成骨细胞MC3T3-E1(3T3)的增殖及成骨分化的作用机制。方法制备羟基磷灰石(HA)和si-HA纳米粒子,以10 mg/L的剂量刺激小鼠RAW264.7巨噬细胞(RAW,分别为HA组和si-HA组),另设Control组(10%FBS的DMEM培养基)。分析RAW细胞的极化状态及炎性因子的表达水平。收集完全培养基、HA和si-HA纳米粒子刺激RAW细胞3 d后获得的上清液,制备RAW条件培养基、HA+RAW条件培养基和si-HA+RAW条件培养基,并培养小鼠3T3前成骨细胞(分别为RAW组、HA+RAW组和si-HA+RAW组),并设空白对照组(完全培养基),检测各组细胞增殖和成骨相关基因[M1极化标志物诱导型一氧化氮合酶(iNOS)、M2极化标志物Arginase基因及促炎、抗炎因子肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-6、IL-10和IL-1ra]的表达水平。结果si-HA组巨噬细胞iNOS、TNF-α、IL-1βmRNA表达水平低于Control组和HA组,Arginase、IL-10和IL-1ra mRNA表达水平高于Control组和HA组(P<0.05);IL-6 mRNA表达水平低于HA组(P<0.05),与Control组差异无统计学意义。si-HA+RAW组3T3细胞增殖能力、碱性磷酸酶(ALP)活性及矿化结节形成明显强于空白对照组、RAW组和HA+RAW组(P<0.05)。培养14 d时,si-HA+RAW组3T3细胞ALP、OCN、OPN、Col-1、Runx2 mRNA表达水平均高于其他3组(P<0.05)。结论si-HA纳米粒子可诱导巨噬细胞产生较有利的骨免疫微环境,增强3T3细胞的增殖和成骨分化。Objective To investigate the mechanism of inorganic bone regeneration material silicon-containing hydroxyapatite(si-HA)in promoting the proliferation and osteogenic differentiation of mouse pre-osteoblast MC3T3-E1(3T3)by regulating the polarity conversion of macrophages.Methods The hydroxyapatite(HA)and si-HA nanoparticles were prepared,and were used to stimulate RAW264.7 macrophages of mice at a dose of 10 mg/L.The cells were divided into HA group and si-HA group.Another control group(DMEM medium with 10%FBS)was set.The polarization status and the expression level of inflammatory factors in RAW cells were analyzed.The supernatants of RAW cells stimulated by HA and si-HA nanoparticles for 3 days were collected to prepare RAW conditioned medium,HA+RAW conditioned medium and si-HA+RAW conditioned medium.The conditioned medium was used to culture mouse 3T3 preosteoblasts,with complete medium as the blank control group.The expression levels of proliferation and osteogenic related genes were detected.Results The results showed that the expression levels of macrophage M1 phenotype factor iNOS,TNF-αand IL-1βmRNA were significantly down-regulated in si-HA group compared with those of the control group and HA group.The expression levels of Arginase,IL-10 and IL-1ra mRNA were significantly higher in si-HA group compared with those of the control group and HA group(P<0.05).The expression level of IL-6 mRNA was lower in HA group(P<0.05).The proliferation ability,alkaline phosphatase(ALP)activity and mineralized nodule formation of 3T3 cells were significantly stronger in si-HA+RAW group than those in the control group,RAW group and HA+RAW group(P<0.05).After 14 days of culture,the expression levels of ALP,OCN,OPN,Col-l and Runx2 mRNA were significantly higher in si-HA+RAW group than those of the other three groups(P<0.05).Conclusion The results show that si-HA nanoparticles can induce a favorable bone immune microenvironment,enhance the proliferation and osteogenic differentiation of 3T3 cells.

关 键 词：巨噬细胞活化 成骨细胞 硅 羟基磷灰石 骨免疫 成骨分化 

分 类 号：R318.08[医药卫生—生物医学工程]