碳-二氧化硅纳米复合颗粒促进小鼠骨髓来源巨噬细胞极化的研究

Effect of carbon-silica nanocomposite on promotion the polarization of mouse bone marrow-derived macrophage

目的研究碳-二氧化硅纳米复合颗粒(carbon-silica nanocomposite,CSN)对M2型小鼠骨髓来源巨噬细胞(bone marrow-derived macrophage,BMDM)极化的影响。方法将介孔二氧化硅纳米复合颗粒(mesoporous silica nanoparticle,MSN)包覆葡萄糖,再经碳化制备得到CSN。利用红外光谱、能量色散X射线谱、透射电子显微镜和扫描电子显微镜对CSN进行表征;CCK-8实验检测CSN对小鼠BMDM的细胞活性的影响;将小鼠BMDM铺于6孔板,设置M0、M1、M2和M2+CSN组,用实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,qPCR)检测M1巨噬细胞中CD86、白介素6(interleukin-6,IL-6)和一氧化氮合酶(nitric oxide synthase,iNOS)及M2型巨噬细胞中CD206、精氨酸酶1(arginase 1,Arg1)的mRNA表达水平;Western blot检测iNOS和Arg1的蛋白表达水平;流式细胞术检测巨噬细胞分子标志物CD80和CD206的表达情况。结果CSN由碳、氧和硅元素组成,元素比例分别为36.6%、35.5%和27.9%,呈均匀分散的球形纳米结构,直径分布为80~90nm。CSN在0、3.125、6.25、12.5、25、50μg/ml浓度下对小鼠BMDM的细胞活性影响无明显差异(P>0.05),与M0组比较,M1组CD86、IL-6和iNOS mRNA表达水平均升高(均P<0.01),iNOS蛋白表达升高(P<0.05),CD80表达升高(P<0.05);M2组CD206和Arg1 mRNA表达水平均升高(P<0.001,P<0.01),Arg1蛋白表达升高(P<0.01),CD206表达升高(P<0.001)。与M2组比较,M2+CSN组CD86、IL-6和iNOS mRNA表达水平均升高(均P<0.001),iNOS蛋白表达升高(P<0.05),CD80表达升高(P<0.05);CD206和Arg1 mRNA表达水平均降低(P<0.05,P<0.001),Arg1蛋白表达降低(P<0.05),CD206表达降低(P<0.05)。结论CSN能促进M2型巨噬细胞向M1型转化,调节肿瘤相关巨噬细胞(tumor-associated macrophage,TAM)中M1/M2比例。

Objective To investigate the effects of carbon-silica nanocomposite(CSN)on the polarization of mouse bone marrow-derived macrophage(BMDM)with M2-type.Method Mesoporous silica nanoparticle(MSN)were coated with glucose and then carbonized to prepare CSN.The CSN was characterized by infrared spectra,energy dispersive X-ray spectrum(EDS),transmission electron microscope(TEM)and scanning electron microscope(SEM).CCK-8 assay was used to detect the effect of CSN on the cell activity of mouse BMDM.Mouse BMDM were plated in 6-well plates,and M0,M1,M2 and M2+CSN groups were set.The mRNA levels of CD86,interleukin-6(IL-6)and nitric oxide synthase(iNOS)in M1 macrophages and CD206 and Arg1 in M2 macrophages were detected by quantitative real-time polymerase chain reaction(qPCR).The protein expression levels of iNOS and Arg1 were measured by Western blot.The expression of CD80 and CD206 in macrophage was detected by flow cytometry.Result CSN was composed of carbon,oxygen and silicon with the proportions of 36.6%,35.5%and 27.9%,respectively.It was a uniformly dispersed spherical nanostructure with a diameter distribution of 80~90nm.CSN concentrations at 0,3.125,6.25,12.5,25 and 50μg/ml had no significant difference in mouse BMDM cell activity(P>0.05).Compared with M0 group,the mRNA expression level of CD86,IL-6 and iNOS in M1 group increased(all P<0.01),the expression of iNOS protein increased(P<0.05),CD80 expression increased(P<0.05).The mRNA expression level of CD206 and Arg1 increased in M2 group(P<0.001,P<0.01),Arg1 protein expression increased(P<0.01),CD206 expression increased(P<0.001).Compared with M2 group,the mRNA expression level of CD86,IL-6 and iNOS increased in M2+CSN group(all P<0.001),as well as the expression of iNOS protein(P<0.05),CD80 expression(P<0.05).However,the mRNA expression level of CD206 and Arg1 decreased(P<0.05,P<0.001),Arg1 protein expression decreased(P<0.05)and CD206 expression decreased(P<0.05).Conclusion CSN can promote the transformation of M2-type macrophage to M1-type,regulate the M1/M2 ratio of tumor-associated macrophage(TAM)。