Kruppel样因子4抑制巨噬细胞向破骨细胞分化介导平滑肌细胞钙化

Kruppel-like factor 4 inhibits the differentiation of macrophages into osteoclasts and mediates calcification of vascular smooth muscle cells

目的探讨Kruppel样因子4(KLF4)在巨噬细胞向破骨细胞分化中的作用及相关机制。方法提取雄性C57BL/6J小鼠腹腔原代巨噬细胞并鉴定,将其分为对照组,NF-κB受体激动剂配体(RANKL)组,共刺激组(KLF4抗体和RANKL共刺激4 d),用抗酒石酸酸性磷酸酶(TRAP)染色检测破骨细胞分化情况,用鬼笔环肽染色检测破骨细胞成熟程度;通过骨吸收陷窝实验测破骨细胞骨吸收能力;通过qRT-PCR检测破骨细胞分化相关基因表达,用钙盐染色检测血管平滑肌细胞钙化情况。结果TRAP染色显示,TRAP阳性细胞直径约100μm,细胞核>3个。共刺激组破骨细胞数量较RANKL组明显增多(P<0.05)。与RANKL组比较,共刺激组破骨细胞成熟程度进一步升高、陷窝面积及数量升高(P<0.05)。RANKL组较对照组相关破骨基因表达明显升高(P<0.05);共刺激组较RANKL组相关破骨基因表达明显升高(P<0.05),而血管平滑肌细胞钙化明显降低(P<0.05)。结论KLF4抗体可促进巨噬细胞向破骨细胞转分化,进而抑制平滑肌细胞钙化。

Objective To study the role of Kruppel-like factor 4(KLF4)in differentiation of macrophages into osteoclasts and its mechanisms.Methods The primary macrophages extracted from the peritoneal cavity of male C57BL/6J mice and identified with optical and CD68 immunofluorescence staining were divided into control group,RANKL group,co-stimulation group,RANKL+anti-KLF4 group.The differentiation of osteoclasts was detected with TRAP staining.The maturity of osteoclast was assayed with phalloidin staining.The capacity of osteoclast bone resorption was tested by bone resorption pit test.The expression of osteoclast differentiationrelated genes was detected by qRT-PCR.The calcification of vascular smooth muscle cells(VSMC)was detected with alizarin red staining.Results TRAP staing showed that the diameter of TRAP positive cells was about 100μm,the number of nuclei are>3.The number of osteoclasts was significantly greater,the maturity of osteoclasts was significantly higher,the pit area was significantly larger,the number of pits was significantly greater,and the expression level of osteoclasts-related genes was significantly higher while the calcification level of VSMC was significantly lower in co-stimulation group than in RANKL group(P<0.05).Conclusion Anti-KLF4 promotes the differentiation of macrophages into osteoclasts by inhibiting the calcification of VSMC.