Li X, Zhang W
Department of Spinal Surgery, Huazhong University of Science and Technology Union Shenzhen Hospital, Shenzhen 518052, China.
First Clinical Medical College of Zhejiang Chinese Medical University, Hangzhou 310059, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2021 Dec 20;41(12):1773-1779. doi: 10.12122/j.issn.1673-4254.2021.12.03.
To explore the mechanism through which tumor necrosis factor-α (TNF-α) promotes osteoclast differentiation.
Bilateral knee joint samples were collected from 4-month-old wild-type mice and TNF-α transgenic mice for CT scan analysis, TRAP staining and sialic acid staining analysis. The osteoclast precursor (RAW264.7) cells were cultured for 3 days in induction medium in the presence of vehicle, TNF-α, or TNF-α and sialidase, and were then examined with RT-qPCR, TRAP staining, and sialic acid immunofluorescence co-localization staining. Bone marrow-derived macrophages isolated from the wild-type mice and TNF-α transgenic mouse and cultured in induction medium with or without the addition of sialidase, and TRAP and sialic acid staining was performed after 3 days of cell culture.
TRAP staining showed that the number of osteoclasts increased significantly in TNF-α transgenic mice as compared with the wild-type mice ( < 0.0001), and micro-CT analysis revealed significant reductions of BV/TV, Tb.N, and Tb.Th in TNF-α transgenic mice ( < 0.001). The osteoclasts in TNF-α transgenic mice also showed a significantly increased expression of sialic acid (=0.004). In the cell experiment, RAW264.7 cells cultured with TNF-α showed a significantly higher expression of sialic acid ( < 0.0001) and a greater osteoclast formation rate (=0.0007) than the the control cells, while the addition of sialidase significantly reduced sialic acid expression, osteoclast formation rate and TRAP mRNA level in TNF-α-treated cells ( < 0.0001). Similarly, in the bone marrow-derived macrophages, sialic acid expression and osteoclast formation rate were significantly increased by incubation with TNF-α ( < 0.0001), but the increments were obviously reduced by addition of sialidase in the medium ( < 0.0001).
TNF-α can promote the differentiation and activity of osteoclasts by increasing the sialylation level in the osteoclasts.
探讨肿瘤坏死因子-α（TNF-α）促进破骨细胞分化的机制。
采集4月龄野生型小鼠和TNF-α转基因小鼠的双侧膝关节样本，进行CT扫描分析、抗酒石酸酸性磷酸酶（TRAP）染色和唾液酸染色分析。将破骨细胞前体（RAW264.7）细胞在含有溶媒、TNF-α或TNF-α与唾液酸酶的诱导培养基中培养3天，然后进行逆转录定量聚合酶链反应（RT-qPCR）、TRAP染色和唾液酸免疫荧光共定位染色检测。从野生型小鼠和TNF-α转基因小鼠分离骨髓来源的巨噬细胞，在添加或不添加唾液酸酶的诱导培养基中培养，细胞培养3天后进行TRAP和唾液酸染色。
TRAP染色显示，与野生型小鼠相比，TNF-α转基因小鼠的破骨细胞数量显著增加（<0.0001），显微CT分析显示TNF-α转基因小鼠的骨体积分数（BV/TV）、骨小梁数量（Tb.N）和骨小梁厚度（Tb.Th）显著降低（<0.001）。TNF-α转基因小鼠的破骨细胞唾液酸表达也显著增加（=0.004）。在细胞实验中，与对照细胞相比，用TNF-α培养的RAW264.7细胞唾液酸表达显著更高（<0.0001），破骨细胞形成率更高（=0.0007），而添加唾液酸酶显著降低了TNF-α处理细胞的唾液酸表达、破骨细胞形成率和TRAP mRNA水平（<0.0001）。同样，在骨髓来源的巨噬细胞中，用TNF-α孵育可显著增加唾液酸表达和破骨细胞形成率（<0.0001），但培养基中添加唾液酸酶可明显降低增加幅度（<0.0001）。
TNF-α可通过提高破骨细胞中的唾液酸化水平促进破骨细胞的分化和活性。
