Mac-1在破骨细胞分化中的作用

Role of Mac-1 in osteoclast differentiation

目的:探究巨噬细胞分化抗原1(Mac-1)分子在核因子κB受体活化因子配体(RANKL)诱导的破骨细胞分化中作用的分子机制。方法:取4周龄C57BL/6J小鼠脾细胞,用RANKL及巨噬细胞集落刺激因子(MCSF)诱导,同时用抗CD11b及抗CD18的特异性抗体进行处理,1周后对细胞核和细胞骨架进行染色;用抗CD11b抗体、干扰CD11b基因的慢病毒及其对照空载病毒处理RANKL诱导的破骨细胞,1周后对细胞进行免疫荧光染色;取4周龄C57BL/6J小鼠脾细胞用RANKL及M-CSF诱导,同时用抗CD11b抗体、干扰CD11b的慢病毒及其对照空载病毒分别进行处理,1周后提取总蛋白进行Western blot检测。结果:CD11b抗体组和双抗体组的多核细胞形成率低于对照组和CD18抗体组,双抗体组和CD11b抗体组之间多核细胞形成率的差异不具有统计学显著性;免疫荧光双标结果显示病毒组的CD11b、Syk及NFATc1表达强度均低于对照病毒组,CD11b抗体组的CD11b、Syk及NFATc1表达强度均低于对照组;Western blot结果显示,CD11b抗体组的CD11b、Syk、NFATc1、c-Fos及p-ERK/ERK水平均低于对照组,病毒组的CD11b、Syk、NFATc1、c-Fos及p-ERK/ERK水平均低于对照病毒组。结论:Mac-1分子中CD11b亚基对破骨细胞分化具有促进作用。该分子通过激活下游Syk通路,上调c-Fos,增加ERK活性,最终上调NFATc1而促进破骨细胞的分化。

AIM：To investigate the function of macrophage differentiation antigen-1 （Mac-1） in receptor acti-vator of nuclear factor-κB ligand （ RANKL ）-induced osteoclast differentiation and the mechanisms .METHODS： The spleen cells were isolated from 4-week-old C57BL/6J mice, and cultured with RANKL, macrophage colony-stimulating fac-tor and CD11b and CD18 antibodies for 1 week.The actin bundles were stained with rhodamine-labeled phalloidin, and nu-clei were stained with DAPI.CD11b and CD18 antibodies, lentivirus with interfering vector plasmid of target gene Itgam （encoding CD11b） and empty virus （control virus） were used to treat osteoclasts for 1 week, and then immunofluorescence staining was performed.CD11b antibody, lentivirus and control virus were used to treat osteoclasts for 1 week, and total protein was taken for Western blot .RESULTS：Lower multinuclear positive rates in the groups treated with CD 11b anti-body were observed than that in the groups treated with CD 18 antibody and control group .Lower immunofluorescence inten-sity of Syk, CD11b and NFATc1 was found in CD11b antibody group than that in control group .Lower Syk, CD11b and NFATc1 immunofluorescence intensity was also observed in lentivirus group than that in control virus group .The results of Western blot analysis showed that the protein levels of CD 11b, Syk, NFATc1, c-Fos and p-ERK/ERK in CD11b antibody group were decreased as compared control group .Compared with control virus group , the protein levels of CD11b, Syk, NFATc1, c-Fos and p-ERK/ERK in lentivirus group were also decreased .CONCLUSION：CD11b subunit of Mac-1 pro-motes osteoclast differentiation by up-regulating c-Fos, ERK activity and NFATc1.